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An Ecologically based study of germination requirements and dormancies in three commercially produced Florida native wil...

University of Florida Institutional Repository

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AN ECOLOGICALLY BASED STUDY OF GERMINATION REQUIREMENTS AND DORMANCIES IN THREE COMMERCIALLY PRODUCED FLORIDA NATIVE WILDFLOWERS By STEVEN MATTHEW KABAT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Steven Kabat

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This document is dedicated to my wife and our parents.

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ACKNOWLEDGMENTS First, I would like to thank the Florida Department of Transportation (FDOT) Environmental Management Office for the generous funds provided for this research. I would additionally like to thank the FDOT and the Florida Federation of Garden Clubs for their integral role in initiating the Florida Wildflower License Plate that allowed the citizens of Florida to voluntarily fund their roadside beautification. I would like to thank my supervisory committee for all of their guidance. I thank Dr. Bijan Dehgan, my supervisory committee chairman, for all his support and encouragement. Without his help and confidence in me, I would not have been able to complete this masters project, and for this I will always be grateful and appreciative. Much more invaluable assistance came from my other committee members, Dr. Jeffrey Norcini and Dr. Doria Gordon. I thank Dr. Jeffrey Norcini for his knowledge of seed biology and his guidance during my pursuit of knowledge. I thank Dr. Doria Gordon for always making time for me in her busy schedule and all her understanding. All my committee members have been extremely willing to help and have enabled me to succeed during my graduate career. I would also like to thank the laboratory crew that made my time more productive and efficient. I thank Fe Almira for all her help in my day-to-day scheduling of activities and her generous, kind nature. I also thank Katherine Turner for her skillful execution of viability testing and seed counting that was tediously performed by hand. I also appreciate her cheerfulness and could not have asked for a better laboratory assistant. iv

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I also thank Fred Bennett for all his help teaching me the workings of the scanning electron microscope. I would also like to thank Marinela Capanu for her voluntary assistance in the statistical analysis of my project. I am so grateful for the love and support of my family, especially my parents, Richard Kabat and Joan Kabat. Both have given me the freedom to make my own decisions and have supported me in those decisions. They have always stressed the value of education and of hard work, and for this I am grateful. I would especially like to thank my mother who has been a wonderful guiding force in my life. I would also like to thank my fatherand mother-in-law Terrell and Colleen Touchton. They have treated me as a son and have supported their daughter and me in all we do. I could not have asked for a better second family. Finally I would like to thank my wife, Cathleen Kabat, who has played a crucial role in my education and research. She has encouraged me to work up to and beyond what I had believed my potential to be. I also thank her for all the time she spent working on this project. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES.............................................................................................................x ABSTRACT......................................................................................................................xii CHAPTER 1 INTRODUCTION........................................................................................................1 Importance of Seed Origin............................................................................................2 Genera...........................................................................................................................4 Coreopsis...............................................................................................................4 Gaillardia..............................................................................................................6 Rudbeckia..............................................................................................................9 Germination and Dormancy in the Asteraceae...........................................................13 Coreopsis Germination and Dormancy Characteristics......................................17 Coreopsis leavenworthii...............................................................................17 Coreopsis basalis.........................................................................................18 Coreopsis bigelovii.......................................................................................18 Coreopsis lanceolata....................................................................................19 Coreopsis palmata........................................................................................22 Coreopsis rosea............................................................................................23 Coreopsis tinctoria.......................................................................................23 Gaillardia Germination and Dormancy Characteristics......................................24 Rudbeckia Germination and Dormancy Characteristics......................................25 2 MATERIALS AND METHODS...............................................................................27 Coreopsis leavenworthii Origin..................................................................................27 Gaillardia pulchella Origin........................................................................................27 Rudbeckia hirta Origin...............................................................................................28 Species Treatment.......................................................................................................28 Statistical Analysis......................................................................................................32 Scanning Electron Micrograph...................................................................................32 3 RESULTS...................................................................................................................34 vi

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2001 Coreopsis leavenworthii....................................................................................34 2002 Coreopsis leavenworthii....................................................................................36 2002 Gaillardia pulchella...........................................................................................39 2002 Rudbeckia hirta..................................................................................................46 4 DISCUSSION AND CONCLUSIONS......................................................................54 Coreopsis leavenworthii.............................................................................................54 Gaillardia pulchella....................................................................................................60 Rudbeckia hirta...........................................................................................................65 Triphenyltetrazolium Chloride Testing Technique....................................................68 Implications for Roadside Plantings...........................................................................69 Coreopsis leavenworthii (mid-June Harvest)......................................................69 Gaillardia pulchella (Late Season harvest).........................................................70 Rudbeckia hirta (Late Season harvest)................................................................70 APPENDIX A Coreopsis leavenworthii FIGURES...........................................................................72 B Gaillardia pulchella FIGURES................................................................................105 C Rudbeckia hirta FIGURES.......................................................................................122 REFERENCES................................................................................................................139 BIOGRAPHICAL SKETCH...........................................................................................143 vii

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LIST OF TABLES Table page 3-1 Main and interactive effects of temperature, light, and months of stratification on germination of viable achenes of C. leavenworthii harvested June 19, 2002..........34 3-2 Main and interactive effects of temperature, light, and months of stratification on germination of viable achenes of G. pulchella harvested August 12, 2002.............35 3-3 Main and interactive effects of temperature, light, and months of stratification on germination of viable achenes of R. hirta harvested August 9, 2002......................35 3-4 Maximum percent germination (n = 4 + SD) of total 2001-harvested C. leavenworthii achenes with statistically insignificant months for each treatment...37 3-5 Maximum percent germination (n = 4 + SD) of total 2002-harvested C. leavenworthii achenes with statistically insignificant months for each treatment.....................................................................................................40 3-6 Percent germination (n = 4 + SD) of freshly harvested and 14-month dry stored (DS) viable C. leavenworthii achenes harvested in 2002.........................................41 3-7 Percent viability (n = 4 + SD) of freshly harvested and 14-month dry stored (DS) C. leavenworthii achenes harvested in 2002.........................................43 3-8 Maximum percent germination (n = 4 + SD) of total 2002-harvested G. pulchella achenes with statistically insignificant months for each treatment..........................44 3-9 Percent germination of viable (n = 4 + SD) freshly harvested and 12-month dry stored (DS) G. pulchella achenes harvested in 2002...............................................46 3-10 Percent viability (n = 4 + SD) of freshly harvested and 12-month dry stored (DS) G. pulchella achenes harvested in 2002...................................................................47 3-11 Maximum percent germination (n = 4 + SD) of stratified and 12-month dry stored (DS) G. pulchella achenes harvested in 2002...............................................48 3-12 Maximum percent germination (n = 4 + SD) of total 2002-harvested R. hirta achenes with statistically insignificant months for each treatment..........................51 viii

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3-13 Percent germination of viable (n = 4 + SD) freshly harvested and 12-month dry stored (DS) R. hirta achenes harvested in 2002 for each treatment.........................52 3-14 Percent viable (n = 4 + SD) of freshly harvested and 12-month dry stored (DS) viable R. hirta achenes harvested in 2002 for each treatment.........................53 ix

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LIST OF FIGURES Figure page 1-1 Scanning electron micrograph of a cross-section of a C. leavenworthii achene at 250X magnification....................................................................................................7 1-2 Scanning electron micrograph of an intact C. leavenworthii achene at 35X magnification..............................................................................................................8 1-3 Scanning electron micrograph of the pappus of an intact C. leavenworthii achene at 150X magnification....................................................................................................9 1-4 Scanning electron micrograph of a G. pulchella achene at 30X magnification of an achene longitudinal-section with the aristate pappus scales present........................10 1-5 Scanning electron micrograph of G. pulchella achene at 40.6X magnification of the exterior of an intact achene with the basal tuft of trichomes evident.......................11 1-6 Scanning electron micrograph of R. hirta achene at 45X magnification of the exterior of an intact achene with evident longitudinal ribbing along the length of the linear cylindrical body...................................................................................12 1-7 Scanning electron micrograph of R. hirta achene at 50X magnification of an achene longitudinal-section with conspicuous embryo............................................13 2-1 Mean minimum, maximum, and monthly temperatures (C) for Gainesville, FL, recorded from 1951 through 1980 (SCS 1985)........................................................30 2-2 Extreme high, extreme low, and monthly mean precipitation (cm) for Gainesville, FL, recorded from 1951 through 1980 (SCS 1985).................................................31 3-1 Germinated R. hirta achenes harvested after 4 months of stratification in their cloth stratification envelope..............................................................................................50 A-1 Mean (n = 4) percent germination (+ SD) of total 2001-harvested C. leavenworthii achenes..............................................................................................72 A-2 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2001-harvested C. leavenworthii achenes at a 12 hr photoperiod.....................................76 x

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A-3 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2001-harvested C. leavenworthii achenes in complete darkness......................................80 A-4 Mean (n = 4) percent germination (+ SD) of total 2002-harvested C. leavenworthii achenes.............................................................................................84 A-5 Mean (n = 4) percent germination (+ SD) of viable 2002-harvested C. leavenworthii achenes.............................................................................................88 A-6 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested C. leavenworthii achenes at a 12 hr photoperiod.....................................92 A-7 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested C. leavenworthii achenes in complete darkness......................................96 A-8 Mean (n = 4) percent viability (+ SD) of 2002-harvested C. leavenworthii achenes............................................................................................100 B-1 Mean (n = 4) percent germination (+ SD) of viable 2002-harvested G. pulchella achenes...................................................................................................................105 B-2 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested G. pulchella achenes at a 12 hr photoperiod..........................................109 B-3 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested G. pulchella achenes in complete darkness...........................................113 B-4 Mean (n = 4) percent viability (+ SD) of 2002-harvested G. pulchella achenes...117 C-1 Mean (n = 4) percent germination (+ SD) of viable 2002-harvested R. hirta achenes..........................................................................................................122 C-2 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested R. hirta achenes at a 12 hr photoperiod.................................................126 C-3 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested R. hirta achenes in complete darkness...................................................130 C-4 Mean (n = 4) percent viability (+ SD) of total 2002-harvested R. hirta achenes......................................................................................................134 xi

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science AN ECOLOGICALLY BASED STUDY OF GERMINATION REQUIREMENTS AND DORMANCIES IN THREE COMMERCIALLY PRODUCED FLORIDA NATIVE WILDFLOWERS By Steven Matthew Kabat August 2004 Chair: Bijan Dehgan Major Department: Environmental Horticulture Three Florida Asteraceae species were examined for one year to determine germination parameters and presence of dormancy. Coreopsis leavenworthii, Gaillardia pulchella, and Rudbeckia hirta achenes were stratified under ambient Florida temperatures or stored dry at room temperature for one year. Achenes were germinated each month at eight different fluctuating temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C) in the light or dark. Germination was recorded at 7, 14, and 21 days, and germinated achenes removed. Nongerminated achenes were tested for viability using 2,3,5-triphenyltetrazolium chloride. Both the June harvested C. leavenworthii and the August harvested G. pulchella achenes exhibited a physiological dormancy followed by a secondary dormancy. Late season harvested R. hirta achenes were nondormant and this was followed by a secondary physiological dormancy. xii

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Coreopsis leavenworthiis primary physiological dormancy was broken after 4 to 6 months of stratification, which resulted in > 96% germination of viable achenes in all but one (23/33C dark) treatment. For the late season harvested G. pulchella, the mild primary physiological dormancy was broken after one month of stratification, which resulted in > 87% germination of viable achenes. After dormancy broke, most G. pulchella achenes became inviable. The achenes that remained viable entered a secondary dormancy (approximately < 15%). After one year of dry storage, germination of viable achenes was > 98%. Rudbeckia hirta achenes harvested in mid-August were nondormant and had > 92% of viable achenes germinate. During the yearlong study, dormancy became strongest in R. hirta after six months of stratification (< 54% germination of viable achenes) followed by a loss of dormancy one year later (> 89% germination of viable achenes). In all three species, dormancy break was characterized by maximum germination of viable achenes, and a rapid germination (within 7 days). xiii

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CHAPTER 1 INTRODUCTION President Clinton issued an Executive Memorandum on Environmentally and Economically Beneficial Landscape Practices on Federal Landscaped Grounds on April 26, 1994 to encourage states to work toward the following goals: use regionally native plants in public landscapes, minimize detrimental effects on natural habitats through environmentally benign construction practices, prevent pollution and misuse of water and energy, and create outdoor demonstration projects to inform the public (Harper-Lore and Wilson 2000). The Florida Wildflower License Plate initiative was implemented to fulfill the directives of the Executive Memorandum. The Florida Department of Transportation (FDOT), in conjunction with the Florida Federation of Garden Clubs, sponsored the Florida Wildflower License Plate, which were to be sold in order to raise the funds necessary for native wildflower research, educational programs, and community-based grant programs. The research component was necessary due to several of the FDOT districts past experiences with planting failures and the lack of technical information about Florida native wildflower seed production. The FDOT realized the need for research focusing on seed germination characteristics, which would provide insight on important aspects of successful stand establishment, including seeding rate, depth of planting, date of seeding, and in what habitats/climates the seeds needed to be planted (Florida Department of Transportation 2003). 1

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2 Importance of Seed Origin When trying to implement native wildflowers into roadside restoration, reclamation and even in commercial and residential landscaping, the utilization of plant material derived from local origins is important. Some sources suggest that plant material should originate from a distance no greater than 160 to 320 km (100 to 200 miles) from the planting location because locally derived plant material is often better adapted to local conditions (greater drought tolerance and increased disease resistance); however, soil, climate, and hydrology may be more important to plant establishment than physical distance (Harper-Lore and Wilson 2000, Pfaff et al. 2002). Non-local plant material may appear more attractive, but some non-local plants may perform poorly over time. Norcini et al. (1998) compared the field performance of Florida native wildflowers Coreopsis lanceolata L., Gaillardia pulchella Foug., and Rudbeckia hirta L. under low input north Florida conditions with seed obtained from commercial producers outside Florida. They found that the north Florida ecotype of C. lanceolata differed morphologically and responded differently than plants from the seed source outside of Florida. The plants grown from seed derived from outside of Florida did not flower at three of the five test sites and also had an increased incidence of disease and insect damage. The G. pulchella plants obtained from different seed sources also exhibited morphological differences. The non-Florida plants flowering duration and overall survival rate was noticeably shorter when compared to the north Florida ecotype. Rudbeckia hirta also exhibited distinct morphological and flowering differences between seed sources (Norcini et al. 1998). It has also been suggested that species inhabiting large geographic ranges and thus exposed to varied climatic conditions may have germination requirements adapted to the

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3 selection pressures of the region of origin (Harper-Lore and Wilson 2000). Another reason to use local ecotypes is to prevent the spread of genetic material (also referred to as genetic pollution) from crops originating from outside the local area or from horticulturally manipulated crops (Pfaff et al. 2002). Wild-crop F1 hybrids of the common sunflower, Helianthus annuus L. (Asteraceae), have changed many population characteristics of the native species (Snow et al. 1998). The cultivated varieties were bred to have strong apical dominance and to be devoid of dormancy. F1 wild-crop hybrids have exhibited decreased flower-head production and branching. These hybrids also have decreased achene production and reduced achene dormancy. Two of the study species, G. pulchella and R. hirta, that are commercially available in Florida are produced in other areas of the country. Both are commonly available in seed catalogs and are used in gardens across the country. Coreopsis leavenworthii Torr. & A. Gray has been available to the public in the large chain stores in Florida since 1999 as part of Riverview Flower Farms Florida Friendly Plants line. In this study, Florida ecotypes of C. leavenworthii, G. pulchella, and R. hirta, were studied in order to determine natural germination parameters and to examine the effect dormancy, if present, had on those germination parameters. This was done by artificial simulation of natural and commercial storage conditions followed by laboratory germination testing at typical Florida seasonal temperatures. Moisture-controlled stratification of achenes was conducted under ambient Florida temperatures in order to simulate the natural environment. The commercial storage technique of a climate-controlled dry storage was also employed. Germination testing followed storage. Dormancy was determined from the characteristics in which germination occurred. The

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4 information obtained was ultimately intended to aid the FDOT in their attempts to employ successful roadside wildflower vegetation management practices. Genera The three genera under study (Coreopsis, Gaillardia, and Rudbeckia) are species-rich. Because of this diversity, historical examination will be limited to germination characteristics within the prescribed genera and to the individual species. Coreopsis Much work has been accomplished with the goal of determining the systematic relationships within the genus Coreopsis (Asteraceae: Tribe Heliantheae: Subtribe Coreopsidinae). There are 75 to 80 recognized species of Coreopsis (tickseed) native to the Americas. The genus Coreopsis is believed to be a paraphyletic assemblage with taxa traditionally prescribed to the genus Bidens arising at two different occasions within the genus Coreopsis. The eastern North American species appear to form a clade that includes, but is not limited to, all the species described below, with the exception of C. bigelovii (A. Gray) H.M. Hall (Kim et al. 1999). The section Calliopsis is composed of three members; C. leavenworthii, C. tinctoria Nutt., and C. paludosa Nutt. Coreopsis leavenworthii appears to be more closely related phylogenetically to C. tinctoria than either one is to C. paludosa (Jansen et al. 1987). Morphological evidence and occupation of the same low-elevation, moist, ruderal habitats suggest the possibility of hybridization between C. leavenworthii and C. tinctoria in northwestern peninsular Florida (Crawford et al. 1984; Smith 1978). These species, however, are believed to retain distinct morphological differences through the majority of their ranges and have not been suggested for species combination (Crawford et al. 1984).

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5 Kim et al. (1999) have proposed that section Eublepharis (C. rosea and C. gladiata Walter) is nested within section Calliopsis (C. leavenworthii, C. tinctoria, and C. paludosa M.E. Jones). The Eublepharis + Calliopsis clade is sister to section Coreopsis (C. basalis [Dietr.] Blake, C. grandiflora Hogg ex Sweet, C. lanceolata, and relatives). Section Palmatae (C. palmata Nutt. and relatives) is a member of a clade that also contains the above listed groups along with section Silphidium and several species of Bidens. Coreopsis bigelovii (Section Pugiopappus) is the most distantly related to C. leavenworthii and is not even included in the eastern North American clade (Kim et al. 1999). In Florida, there are 13 species of Coreopsis found in the wild. Of these, C. floridana E.B. Sm. and C. leavenworthii are endemic, and some scientists believe C. basalis and C. tinctoria have naturalized in Florida from other areas of the United States (Wunderlin and Hansen 2003). Coreopsis leavenworthii (Leavenworth's tickseed) is an herbaceous annual to short-lived perennial. Its flowers are aggregated into capitate heads that are less than 5 cm in diameter on long peduncles in open corymbs (Ledin 1951). The heads are composed of sterile yellow ray florets and fertile dark brown disk florets. The floral heads are observable from mid-spring to fall in northern Florida to all year in southern Florida, but are particularly abundant in spring (Taylor 1998). The fruit is a thin oval achene (Fig. 1-1) with two tan lateral wings. Each wing is as broad as the achenes dark brown body, which contains the embryo (Fig. 1-2). The achene also has a pappus of two short awns (Fig. 1-3) (Ledin 1951).

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6 Coreopsis leavenworthiis range covers most of Florida, but has not been vouchered in Calhoun, Clay, Escambia, Gulf, Hamilton, Holmes, Leon, Liberty, Madison, Nassau, or Okaloosa Counties, probably due to under-collection. It has been documented in floras as far back as 1887 (Chapman 1887). The oldest herbarium specimen at the University of Florida is from Tampa, Hillsborough County in 1876. Coreopsis leavenworthii grows in wet pine flatwoods, glades, prairies, and various ruderal sites, including roadside ditches and, rarely, in dry pinelands (Ledin 1951; Taylor 1998). Pine flatwoods, which comprise 50 percent of the land area in Florida, are characterized by low, flat topography and sandy, acidic soil underlain by a clay hardpan (which restricts drainage and often creates a seasonally high water table). Prior to European settlement, frequent fires naturally maintained pine flatwoods. The largest diversity of Florida wildflowers occurs in open-canopy flatwoods where fire is frequent (Myers and Ewel 1990). Gaillardia The genus Gaillardia (Asteraceae: Tribe Heliantheae) is represented in Florida by two species: G. aestivalis (Walter) H. Rock and G. pulchella. Gaillardia pulchella is commonly known as Blanket Flower, Indian Blanket, or Firewheel (Turner and Whalen 1975; Wunderlin and Hansen 2003). It has been documented nearly throughout Florida and is often used as an ornamental that tends to freely reseed outside of its original planting site (Bell and Taylor 1982; Turner and Whalen 1975; Wunderlin and Hansen 2003). The species range covers most of the United States, excluding the northwestern states, west of South Dakota and Minnesota (Niering and Olmstead 2001). Gaillardia pulchella is a halophyte (salt-loving plant) but is not limited to coastal sites, also

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7 Figure 1-1. Scanning electron micrograph of a cross-section of a C. leavenworthii achene at 250X magnification. The cross-section was made perpendicular to the achenes wings. The bar in the figure is equivalent to 0.12 mm. The image was recorded on a Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology Research in the Electron Microscopy Core Laboratory, University of Florida. inhabiting prairies, sandy open sites, and roadsides (Niering and Olmstead 2001). In coastal ecosystems, G. pulchella contends with shifting sand, sand abrasion, intense sunlight, nutrient-poor soils, salt spray, and desiccation (Taylor 1998). Turner and Whalen (1975) recognize three varieties (picta, australis, and pulchella), but I will refer to the species as a whole, as per Wunderlin and Hansen (2003). Gaillardia pulchella is an annual to short-lived perennial. Its peak bloom time in Florida occurs from May through August, but bloom may occur any time of year (Wunderlin and Hansen 2003). Flowerheads are born terminally, with 6 to 15 ray florets. The ray florets

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8 Figure 1-2. Scanning electron micrograph of an intact C. leavenworthii achene at 35X magnification. The achenes two prominent wings are evident. The bar in the figure is equivalent to 1 mm. The image was recorded on a Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology Research in the Electron Microscopy Core Laboratory, University of Florida. may be entirely red, entirely yellow, or reddish purple with yellow notched apices (Bell and Taylor 1982). The center of the head is composed of fertile reddish purple disk florets. Floral heads range from 2.5 to 7.5 cm in diameter (Taylor1998). Achenes are born on a receptacle containing firm, subulate (awl-shaped) setae. Achenes have 6 to 10 aristate (stiff apical bristle or awn) pappus scales (Fig. 1-4) and a conspicuous basal tuft of trichomes (Fig. 1-5) (Turner and Whalen 1975; Wunderlin and Hansen 2003). Gaillardia pulchella is a diploid, obligate outcrosser (self-incompatible) (Heywood 1993).

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9 Figure 1-3. Scanning electron micrograph of the pappus of an intact C. leavenworthii achene at 150X magnification. The achenes pappus is composed of two short awns. The bar in the figure is equivalent to 0.2 mm. The image was recorded on a Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology Research in the Electron Microscopy Core Laboratory, University of Florida. Rudbeckia The genus Rudbeckia is in the Helianthus tribe in the Asteraceae. There are about 25 species native to North America. There are nine Rudbeckia taxa in Florida, including R. hirta, commonly known as Black-eyed Susan. Rudbeckia hirta occurs throughout North America, except for Arizona, Nevada, and the far north (Harkess and Lyons 1994). In Florida, R. hirta commonly grows in sandhills, pine flatwoods, and open disturbed sites and is found nearly throughout the state (Wunderlin and Hansen 2003). Rudbeckia hirta is an annual to short-lived perennial. In Florida, flowering primarily occurs from April through October. The flowerheads contain yellow ray florets

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10 Figure 1-4. Scanning electron micrograph of a longitudinally sectioned G. pulchella achene at 30X magnification with the aristate pappus scales present. The bar in the figure is 1 mm. The image was recorded on a Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology Research in the Electron Microscopy Core Laboratory, University of Florida. and purple brown disk florets that are attached to a chaffy conical receptacle. The disk florets are fertile and have divided styles (Harkess and Lyons 1994; Niering and Olmstead 2001; Norcini personal communication). The actinomorphic heads measure 3.8 to 7.5 cm across (Niering and Olmstead 2001; Norcini et al. 2001). Achenes are dark brown and linear with longitudinal ribbing along the length of the cylindrical body (Fig. 1-6) that contains the embryo (Fig. 1-7). As was mentioned in the species description, R. hirta covers much of the eastern United States. Marois and Norcini (2003) investigated regional differences in the species in a field trial conducted in Quincy, Florida. They found significantly lower season-long

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11 Figure 1-5. Scanning electron micrograph of the exterior of a G. pulchella achene at 40.6X magnification with the basal tuft of trichomes evident. The bar in the figure is 1 mm. The image was recorded on a Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology Research in the Electron Microscopy Core Laboratory, University of Florida. survival from a Texas source as compared to those attained from northern Florida and central Florida seed sources, although they could not attribute the difference in plant survival to fungal attack, irrigation, or climate (precipitation or temperature). Consequently, seed source was the only attributable difference, but they recognized the need for further study into the differences in survival. It was also hypothesized that the cause could have been due to the Texas material coming from a shorter-season population as compared to Florida (Marois and Norcini 2003). Another study by Norcini et al. (2001) examined latitudinal site variability (four sites within Florida) on plant growth, flowering, and survival among two Florida ecotypes and one from Texas.

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12 Figure 1-6. Scanning electron micrograph of an intact R. hirta achene at 45X magnification with evident longitudinal ribbing along the length of the linear cylindrical body. The bar in the figure is 1 mm. The image was recorded on a Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology Research in the Electron Microscopy Core Laboratory, University of Florida. Despite similarities in the soil texture, the sites had different rainfall amounts, nutrient levels, and degrees of nematode infestations. Collectively, the environmental differences somewhat obscured the results. Survival was significantly higher for the Florida ecotypes in three of the four sites compared to the Texas ecotype, despite site variability. The fourth site showed no variability among ecotypes, but the plants had an unusually short lifespan compared to the other sites (Norcini et al. 2001). Rudbeckia hirta has been used as a model species in the study of wildflower sod production. Neigebauer et al. (2000) examined three mowing heights, 5.1 cm, 7.6 cm

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13 Figure 1-7. Scanning electron micrograph of a longitudinally sectioned R. hirta achene at 50X magnification with the conspicuous embryo present. The bar in the figure is 1 mm. The image was recorded on a Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology Research in the Electron Microscopy Core Laboratory, University of Florida. (the height typically used in wildflower sod production), and 10.2 cm, and compared them to plant material that was not mowed. The study concluded that, as mowing height was increased from 5.1 cm to a non-mowed condition, there was a corresponding increase in root dry-weight, rooting depth, and the amount of root axes in the top 2.5 cm of soil, the layer typically harvested in production. Germination and Dormancy in the Asteraceae Nondormant seeds that do not germinate for reasons other than a lack of sufficient external moisture or unfavorable environmental conditions are not necessarily dormant, they may just be quiescent. Quiescent seeds sometimes have been referred to as having an enforced dormancy or said to have an environmental inhibition to germination (Baskin

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14 and Baskin 2001). An environmental factor such as available moisture or oxygen (excluding seeds physical structure) able to be changed may enhance germination by ending quiescence. Quiescent seeds are non-dormant seeds in which metabolism is reduced or halted due to an environment that is below or above that required for optimal germination. Dormant seeds are unable to germinate even after exposure to optimal germinating conditions, while quiescent seeds will germinate upon exposure to optimal conditions (Baskin and Baskin 2001). In addition, dormant seeds have one or more barriers to overcome before germination can occur and these barriers collectively make up a seeds dormancy (Baskin and Baskin 2001). Primary (innate or conditional) dormancy occurs when seeds are dormant directly after they are released from the mother plant. Physiological dormancy (PD) is a type of primary dormancy and is endogenous or embryo-based with germination inhibited by a physiological mechanism. There are three types of PD: nondeep, intermediate, and deep. Nondeep PD is characterized by recently matured seeds that only germinate over a narrow temperature range (referred to as conditional dormancy or relative dormancy) or by a lack of germination at any temperature (primary innate dormancy). Seeds with nondeep PD are the only seeds that have the ability to annually cycle between dormancy and nondormancy. Such seeds go through a state of conditional dormancy during the transitional period. This cycling is gradual and occurs in response to environmental cues. Some seeds only cycle between conditional dormancy and nondormancy (Baskin and Baskin 2001; Bewley and Black 1994). The literature refers to afterripening in two different ways. It is refers to as the process dry stored seeds undergo as they become nondormant and it is also referred to as

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15 the duration storage (typically dry) as a dormancy breaking technique (Baskin, Baskin, and Van Auken 1992; Bewley and Black 1994). The latter definition will be used in this document. Afterripening occurs in dry stored seeds, but the seeds themselves may have up to 20% water content. There are three different response patterns associated with dormancy break. During the type-1 response, the maximum temperature limit at which germination may occur increases as conditional dormancy is broken. In a type-2 response, a seeds minimum temperature at which germination may occur decreases. The type-3 response is characterized by both the upper and lower germination temperature limits increasing and decreasing, respectively. Of the 32 Asteraceae species whose response process had been studied by Baskin, Baskin, and Van Auken (1992), three were type-1, 22 were type-2, and seven were type-3. The type-1 response pattern is usually found in species with a winter life cycle. Both the limited number of species studied and, possibly, the small number of species that experience a winter life cycle may be the hypothesized explanation for the limited number of species encountered with the type-1 response pattern (Baskin, Baskin, and Van Auken 1992). Secondary (induced) dormancy occurs when mature imbibed seeds experience sub-optimal or super-optimal environmental conditions (anaerobic environment, too much darkness or light, temperatures excessively high or low, or water stress) that trigger dormancy. Similar to primary dormancy, secondary dormancy may be innate or conditional. Secondary dormancy often begins as a seed coat imposed dormancy and then develops into a condition of embryo dormancy (Baskin and Baskin 2001; Bewley and Black 1994).

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16 Another sign that seeds may have a nondeep PD is the presence of a light requirement that may or may not be lost as dormancy is broken (Baskin and Baskin 2001). Also, seeds that have a nondeep PD can achieve normal embryo growth when the embryo is isolated from the seed. Barriers associated with nondeep PD are usually attributed to the interaction between the embryo and its covering structures. The covering structures may influence dormancy by: controlling oxygen passage to the embryo, controlling the movement of growth inhibitors away from the embryo, or physically restricting the embryos growth potential by controlling enzymatic breakdown of embryo covering structures. Nondeep PD may be broken through the application of exogenous chemicals, including kinetin, gibberellins, ethylene, and potassium nitrate (KNO3), that effectively substitute for specific environmental dormancy breaking conditions (Baskin and Baskin 2001). An intermediate PD is characterized by the need for an increased duration (< 6 months) of dormancy-breaking treatment (mainly stratification) prior to germination. The intermediate PDs dormancy-breaking treatment may be shortened by room-temperature dry storage or by the application of exogenous chemicals. Isolated embryos with an intermediate PD can also achieve normal embryo growth upon removal from the seed like that of a nondeep PD. Deep PD is differentiated by the requirement of a long period of dormancy-breaking treatment for intact dispersal units; it cannot be shortened by implementing any of the aforementioned techniques and isolated embryos do not develop normally (Baskin and Baskin 2001).

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17 There are various other types of dormancies that may occur in combination with physiological dormancy. When time is required for growth of an underdeveloped or undifferentiated embryo prior to germination, it is a type of endogenous dormancy termed morphological dormancy (Baskin and Baskin 2001). Physical dormancy is the result of a water impermeable fruit or seed coat. Chemical dormancy prevents or inhibits germination by chemical inhibitors usually located in the pericarp. The inhibitors need to be leached or physically removed before chemical dormancy is broken and germination can occur. Abscisic acid is commonly associated with chemical dormancies. However, it is not known to what degree it is responsible for the cause of a chemical dormancy. Mechanical dormancy (coat-imposed dormancy) is a restriction of growth due to a woody fruit wall usually formed from the endocarp and/or mesocarp or the endosperm. In a mechanical dormancy the surrounding structures restrict growth of the embryo until dormancy is broken. All of the above types of dormancies may occur separately or in combination with one another. Many members of the Asteraceae experience physical dormancy due to a semi-permeable inner membranous seed coat that has the ability to retard water and oxygen exchange with the embryo. The semi-permeable membrane may also prevent leaching of germination inhibitors located within the cotyledons (Atwater 1980). Some species of Asteraceae, including tickseeds (Coreopsis), also may have a waxy covering on the seed coat that may prevent water and/or gas exchange (Voigt 1977). Coreopsis Germination and Dormancy Characteristics Coreopsis leavenworthii Beyond the morphological species description, distribution range in Florida, and growth habitats, little information is available about C. leavenworthii, especially

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18 regarding its biology or ecology. Norcini and Aldrich (2003) examined sowing date on six Florida wildflowers. Plots (15 ft2) were sown with 2001 and 2002 harvested C. leavenworthii achenes during the first week of each month from July through December 2002 and evaluated on March 24, 2003. The largest number of plants was achieved from the September sowing during both harvest years. The September sowing also visually rated well when judged on flowering, wildflower density, and weediness. The more recently harvested 2002 achenes had equivalent or higher number of established plants throughout testing. The 2002 harvest also had good plant establishment between August and November. Coreopsis basalis Coreopsis basalis is found from Floridas central peninsula north into the panhandle in scattered areas (Wunderlin and Hansen 2003). The Association of Official Seed Analysts (AOSA) standardized germination test for C. basalis is performed by placing the achenes on top of a blotter at a constant 20C and checking at 8 days (AOSA 1994). Coreopsis bigelovii Coreopsis bigelovii is a Mojave Desert annual that, in its native environment, exhibited dormancy after dispersal from the mother plant. Capon and Van Asdall (1967) used pre-germination heat treatment to break dormancy; greatest germination (~30%) was attained at 20C after 8 weeks or at 50C after 5 weeks, although a storage time of 1 week at 50C yielded 26% germination. Germination of freshly harvested seed was only 9% (Capon and Van Asdall 1967).

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19 Coreopsis lanceolata In Florida, C. lanceolata can be found growing in sandhills and disturbed sites from northern Florida southward to Lake County (Wunderlin and Hansen 2003). Its broad geographic range extends west to Texas and New Mexico, and north to Lake Superior (Banovetz and Scheiner 1994a; Banovetz and Scheiner 1994b). Germination tests for C. lanceolata have been standardized, but recommended guidelines for optimum germination practices vary throughout the literature. For the AOSA protocol (1994), achenes are germinated on top of a blotter at an alternating 20/30C (or at a constant 15C) in light and germination is checked at 7 and 21 days. The blotters should be moistened with a 0.2% solution of KNO3. Light should be provided during the high temperature and for a minimum of 8 hr. The lower temperature should be held constant for 16 hr followed by 8 hr at the higher temperature (AOSA 1994). Atwater (1980) found that immersing achenes in water followed by immersion in 0.2% KNO3 improved germination at 15C for 40 days. Carpenter and Ostmark (1992) tested the germination characteristics of achenes collected from north-central Florida in May when the achenes were naturally dispersing. Germination tests were conducted at a constant 15C on a double layer of filter paper substrate in Petri dishes wetted with distilled water. The 15C optimal temperature for germination tests was determined after comparison between different constant and alternating temperatures. The effect of growth regulators was also studied. A 6 hr treatment of 1,000 ppm ethephon plus 1,000 ppm gibberellic acid (GA3) significantly improved germination of freshly harvested achenes. After 21 days germination test, 79% of the seeds germinated, with half of them germinating within 6.1 days of the start of the test and the majority of them over a 5.5-day period. Single ethephon and GA3 growth regulator treatments also significantly

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20 improved germination over the control but not to the same degree as the combination treatment. The duration of fresh seed storage was also examined for different relative humidities. Optimal germination (80%) was attained at 15C after 7 months of storage at 20 to 35% relative humidity (Carpenter and Ostmark 1992). Banovetz and Scheiner (1994a) determined that optimal germination could be attained at 15 or 25C under a 12 hr photoperiod. Banovetz and Scheiner (1994a) also reported that germination percentages of C. lanceolata increased as the dry achenes aged from 2 to 20 months; the percentages declined thereafter. This increase in germination percentage was probably a result of afterripening. This primary (innate) dormancy is usually attributed to achene structural or physiological barriers. Coreopsis lanceolata was induced into a secondary dormancy when previously dry-stored achenes were imbibed and placed at 5C for increasing lengths of time and then moved to 24 hr of light. As exposure time at 5C increased, subsequent germination percentage decreased, indicating the existence of a secondary dormancy. The dry-stored achenes that entered into the secondary dormancy could not be brought out of it even with subsequent light/dark cycles, freezing temperatures, or warmer temperatures (Banovetz and Scheiner 1994a). The effect of vegetation cover has been studied with respect to the percentage germination of C. lanceolata in the field. It was concluded that seedlings emerged in significantly higher numbers in non-vegetated as compared to vegetated plots (Banovetz and Scheiner 1994a). The effect of seed mass on the ability to germinate and subsequent seedling viability of C. lanceolata has also been investigated. Achenes of C. lanceolata that had a

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21 greater mass had higher viability after burial and emerged from greater depths as compared to achenes with lower masses. When predispersed achenes of a Michigan ecotype were collected and analyzed, they displayed a traditional bell-shaped curve for seed mass with a mean around 0.8 mg. However, when collected from a native soil seed bank (the majority from 0 to 2 cm in depth), the mean of the majority of achenes was below 0.4 mg (Banovetz and Scheiner 1994b). The authors suggested that this low weight could be attributed to a combination of predation of larger achenes, germination out of the seed bank, and the lower probability that larger-sized achenes can work their way into the seed bank. When the natural seed bank was tested for viability, only those achenes greater than 0.4 mg were viable. While those achenes in the seed bank less than 0.4 mg were almost 30 times more abundant, they were not viable (Banovetz and Scheiner 1994b). As part of the same study, C. lanceolata achenes with a larger mass that were manually buried for two years in the field (i.e., forced into the seed bank) retained their viability longer than achenes with a lower mass. Field-buried achenes with a larger mass, however, displayed lower germination rates, indicating that they had developed dormancy. The dormancy was most likely a secondary dormancy caused by the cold winters in Michigan (Banovetz and Scheiner 1994a). The effect of seed priming on C. lanceolata achenes has also been studied. Osmotic priming (osmoconditioning) is a process that controls or limits imbibition through the use of inorganic salts (e.g., potassium phosphate) or polyethylene glycol (PEG). This controlled imbibition enhances seed viability, improves germination speed and uniformity, and expands the range of temperatures suitable for germination of aged seed by slowing germination processes. The success of osmoconditioning is likely

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22 accomplished by allowing repair of age-induced damage (Bewley and Black 1994; Fay 1994; Samfield et al. 1991). Primed achenes of C. lanceolata exhibited increased germination percentages, rapidity, uniformity, and, ultimately, a faster growing, more uniform crop after priming in a 50 mM potassium phosphate buffer solution and to a slightly lesser extent after priming in distilled water. A 3and 6-day priming treatment at approximately 16C in both osmotica resulted in the greatest priming benefit (Samfield et al. 1991). Achenes of C. lanceolata also responded favorably to 2 months of vacuum storage at 12C. The rate of germination and the total germination percentage was improved over the control that was not vacuum stored. The vacuum probably reduced respiratory activity in storage, resulting in prolonged viability (Samfield et al. 1990). Coreopsis palmata Coreopsis palmata was collected for a study from a northern Illinois prairie remnant (Voigt 1977). The original seed had only 50% embryo development, but of those that developed, 96% tested viable when a 2,3,5-triphenyltetrazolium chloride (TZ) test was administered. The achenes in the experiment were treated with a fungicide (Arasan) and then germinated on moist filter paper in Petri dishes in darkness at room temperature (~24C), although the darkness was interrupted daily for germination counts. Achenes were probably dormant since germination was only 40% after 30 days. Germination increased to 98% after cold stratification at 4C for 60 days. Stratification also narrowed the timing of germination. Germination occurred between 4 and 30 days without stratification and between 6 and 12 days with stratification.

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23 Coreopsis rosea Coreopsis rosea is an obligate perennial (requires two growing seasons to reproduce) found in southeastern Canada south to Georgia. The achenes collected from this region were cold stratified at 4C for 270 days and then germinated in Petri dishes at an alternating temperature of 20/30C and a 15 hr photoperiod. The achenes began to germinate after 6 days and achieved a maximum of 50% germination after 30 days (Shipley and Parent 1991). Coreopsis tinctoria Coreopsis tinctoria is native to the southeastern United States, but is considered a non-native cultivated introduction to Florida by many botanists. It may be found growing in wet and open disturbed areas in the state (Wunderlin and Hansen 2003). The species was not recorded as inhabiting Florida in Chapmans flora (1887) and University of Florida herbarium specimens only date back to a 1932 Alachua County specimen and a 1948 Dade County specimen. Some roadsides in northern Florida have been intentionally seeded with this species by the FDOT and possibly other agencies responsible for roadside vegetation management. The species is regarded as a winter annual weed in temperate grasslands (Baskin and Baskin 2001). The fruit and seed structures have been investigated in great detail (Pandey and Singh 1982). The AOSA protocol (1994) for C. tinctoria requires achenes to be placed on top of a blotter at a constant 20C and germination to be recorded at 8 days. However, Kaspar and McWilliams (1982) reported that optimal germination of C. tinctoria was at constant 30C under continuous illumination. They did not find dormancy in C. tinctoria as evidenced by a pre-study germination percentage of 94%. Total germination (~90%) over a 14-day period was equivalent at constant temperatures of 20, 25, or 30C, but the

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24 treatment at 30C had the most rapid rate of germination, reaching approximately 75% after 2 days. Baskin and Baskin (2001) recommend a 15/25C alternating temperature regime in light (Baskin and Baskin 2001). Meyer Elliott (1999) broke a mild primary physiological dormancy after 3 months of dry storage at 25C and achieved 99+ 0.5% germination. Achenes remained nondormant after 3 months until the end of testing (7 months) (Meyer Elliott 1999). Gaillardia Germination and Dormancy Characteristics The AOSA (1994) protocol for testing achenes of G. pulchella Foug. var. picta (Sweet) A. Gray is to place achenes on top of a blotter at an alternating 20/30C temperature plus light during the warm temperature, then recording germination at 4 and 10 days. Light should be provided by a cool-white fluorescent source for a minimum of 8 and a maximum of 16 hr with an intensity of 750 to 1250 lux (75 to 125ft-c), which is comparable to a fluence rate of 20 mol m-2 sec-1 or 0.29 to 0.48 mol m-2 day-1. The light requirement may be satisfied through the use of one or two 20-W cool white bulbs 15-20 cm above the seeds (AOSA 1994; Baskin and Baskin 2001). Atwater (1980) also tested G. pulchella var. picta and achieved 70% germination when achenes were tested at a 20/30C alternating temperature for 14 days. Baskin et al. (1992) conducted a study to examine the effect an alternating wet/dry storage treatment (simulated natural conditions) would have on dormant G. pulchella achenes. Storage was performed by placing achenes on a sand substrate in Petri dishes that were allowed to dry prior to rewetting. Germination tests were conducted by moving the storage dishes into one of six alternating temperature regimes (6/15C, 10/20C, 15/25C, 15/30C, 20/35C, and a simulated Texas regime normal for the given month of testing). Germination tests were performed under a 14-hr photoperiod for 15 days. After

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25 4 months of storage, dormancy was broken at 10/20C, 15/25C, and 15/28C (simulated Texas Oct. temperature) resulting in > 90 + 3% germination. They also examined achene burial (stratification) over 4 months in moist sand at five different temperature regimes (5C, 6/15C, 10/20C, 15/25C, and 15/30C). Germination tests were performed as above including the simulated Texas temperature regime for October (15/28C). Dormancy only broke after a 15/25C stratification followed by a germination test at 10/20C (90 + 3%) and after a 15/30C stratification followed by a germination test at 6/15C (100%), 10/20C (100%), or 15/25C (84 + 1%). Gaillardia pulchella achenes exhibited an afterripening type-1 response pattern, which was a first for a winter annual Asteraceae (Baskin et al. 1992). Norcini and Aldrich (2003) examined how sowing date affected the establishment of a Florida ecotype of G. pulchella in northern Florida. Plots (15 ft2) were sown with 2002 harvested achenes during the first week of each month from July through December 2002 and evaluated on March 24, 2003. The largest number of plants was achieved from the July sowing. The July sowing also visually rated well when judged on flowering, wildflower density, and weediness. Rudbeckia Germination and Dormancy Characteristics The AOSA protocol (1994) for R. hirta requires achenes to be placed on top of a blotter at an alternating 20/30C in light and germination recorded at 7 and 14 days. Atwater (1980) tested a Rudbeckia species and achieved 85% achene germination when tested at a 20/30C alternating temperature for 14 days. Osmotic priming (osmoconditioning) of Rudbeckia has also been investigated. Priming in an aerated KNO3 solution (osmotic potential .3 MPa) at 30C for 7 days of

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26 a related species, Rudbeckia fulgida Ait., increased the germination percentage and germination speed as compared to non-primed seeds (Fay 1994). An early winter (Nov. or Dec.) seeding date is recomended for R. hirta in Florida so that seeds may be produced by the following summer (Norcini et al. 1999, Pfaff et al. 2002). Norcini and Aldrich (2003) examined how sowing date affected the establishment of a Florida ecotype of R. hirta in northern Florida. Plots (15 ft2) were sown with 2002 harvested achenes during the first week of each month from July through December 2002 and evaluated on March 24, 2003. The largest number of plants was achieved from the September sowing. The September sowing also visually rated the best when judged on flowering, wildflower density, and weediness. Good plant establishment, however, also occurred during October and November. Sowing of achenes is discouraged from March through April because of the tendency for dry and/or windy conditions in upland sites (sandhills) during this time (Pfaff et al. 2002). A thin (7 mm or less) cover of soil is also recommended after sowing (Norcini et al. 1999).

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CHAPTER 2 MATERIALS AND METHODS Germination characteristics of C. leavenworthii, G. pulchella, and R. hirta, were determined for freshly harvested achenes that were buried in containers, subjected to ambient Florida temperatures, and simulated natural moisture conditions. The mass of all achenes was estimated by the mean of 10 replicates of 100 achenes. Coreopsis leavenworthii Origin Coreopsis leavenworthii achenes were obtained from Melton Farms located near Dade City (Pasco County, FL). Melton Farms received their achenes from a restoration site in Polk County, FL, in 2000. The Polk County restoration site was planted with achenes originating from a site in southwest Orlando (Orange County, FL). Achenes were harvested on June 19, 2001 and June 19, 2002. Plants at Melton Farms were grown in rows and provided sub-irrigation once a week. Rows were formed by a 5.1 to 7.6 cm gap left between two parallel strips of landscape fabric. Achenes were vacuumed off the fabric and cleaned at Melton Farms. Testing was started on July 4, 2001 and June 24, 2002 for the 2001 and 2002 harvests, respectively. Gaillardia pulchella Origin Gaillardia pulchella achenes used in all studies were obtained from a first-generation crop grown at Melton Farms that originated from a wild coastal population in northern Daytona (Volusia County, FL). Preliminary testing was conducted on naturally dispersed achenes harvested July13, 2001, August 15, 2001, and October 26, 2001. Heads of achenes and naturally dispersed achenes were harvested on August 12, 2002 for 27

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28 the main study. To have enough achenes for the main study, a portion of the achenes had to be manually removed from their receptacles and combined with achenes that had naturally dispersed. Testing was started on August 20, 2002. Rudbeckia hirta Origin The R. hirta achenes originated from a site in Polk County, FL. Achenes were produced, harvested, and cleaned at Woodhaven Farms in Havana (Gadsden County, FL) on August 9, 2002. Testing was started on August 21, 2002. Species Treatment Employing methods similar to those suggested by Baskin and Baskin (2001), all germination testing was initiated within two weeks of harvest. A small study was conducted in 2001 exploring the effect of time of harvest on viability and germinability. Replicates (n = 8) of 50 G. pulchella achenes were tested at a 20/30C alternating temperature regime on top of blotter paper with a 12-hr photoperiod (AOSA 1994). Germination was recorded at 4 and 10 days. On July 13, 2001, in addition to the methods above, a concurrent test with replicates (n = 3) of 50 achenes was also run using 0.2% KNO3 in place of distilled water as the wetting agent. During the main study, the time of stratification and/or light requirement needed to break dormancy, the speed of germination, and the change in viability and germinability as the length of stratification was increased were examined. The C. leavenworthii studies were approximately one year each for two consecutive years (harvested in 2001 and 2002) while the G. pulchella and R. hirta studies were for one year each (harvested in 2002). Germination treatments were performed each month of stratification (0 to 11 months and 0 to 9 months, respectively, for 2001 and 2002 harvested C. leavenworthii

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29 achenes; 0 to 11 months for G. pulchella and R. hirta). The effect dry-storage had on viability and germinability approximately 1 year after dispersal was also examined. In the main study, over 3,200 achenes of C. leavenworthii and G. pulchella were placed into individual 11 x 14 cm perforated plastic pollination bags for each month of stratification and closed with a drawstring. Due to the smaller achene size of R. hirta, achenes were placed in 5 x 7 cm sheer cloth envelopes fastened with staples. The C. leavenworthii, G. pulchella, and R. hirta achenes required for each month of stratification were 0.75 g, 5.00 g, and 1.08 g, respectively. Each bag of achenes was placed on a 7 cm bed of sand in a standard 3.8-liter black nursery container. Sand was added to containers until each bag was covered by 7 cm of sand. To prevent the loss of sand through the drainage holes, each containers exterior was covered with greenhouse ground cloth and placed into another 3.8-liter container. Each container was initially saturated to field capacity with distilled water and placed on the ground-cloth floor of a plastic-covered shade house (30% shade). The shade house had retractable plastic sides that maintained the temperature at the ambient temperature at ground level. The average temperature in Gainesville, FL, ranges from 7 to 33C (Fig. 2-1). Containers were watered once a week with 450 ml of distilled water, which is equivalent to 2.5 cm of water per week (Fig. 2-2). One packet of achenes was removed from its stratification container each month and placed on a pill-counting plate. Masses of achenes were teased apart to aid drying, which occurred over the subsequent 24 hr. This process was repeated every 3 to 4 weeks. Dry achenes were counted into 50-seed replicates and placed into 64 test tubes. The 100 x 15 mm plastic Petri dishes had previously been prepared by filling with an average of 62 grams of sand (Standard Sand and Silica Co. 12\20 grit) equivalent to a full, level 30 ml

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30 05101520253035JASONDJFMAMJMonthTemperature (C) Monthly maximum mean Monthly mean Monthly minimum mean Figure 2-1. Mean minimum, maximum, and monthly temperatures (C) for Gainesville, FL, recorded from 1951 through 1980 (SCS 1985). glass beaker. Each Petri dish of sand was thoroughly moistened with approximately 13 ml distilled water. The 50 achenes were spread as evenly as possible over the moistened sand. Four dishes per species per incubator were wrapped in plastic film to prevent water evaporation and another four were covered with one layer of aluminum foil to prevent light exposure during germination testing. Eight incubators were programmed to alternate between two temperatures to simulate maximum and minimum daily average temperature fluctuations in an average Florida year (Fig. 2-1). The eight temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C) were based on Florida climate data. The 7-W (Bulbrite Energy Wiser DECO, Moonachie, NJ) soft-white compact florescent lights were on a 12 hr cycle coinciding with the warmer temperature of any given regime. Thermostats were accurate to within 2C.

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31 051015202530354045JASONDJFMAMMonthPrecipitation (cm) J Monthly maximum mean Monthly mean Monthly minimum mean Figure 2-2. Extreme high, extreme low, and monthly mean precipitation (cm) for Gainesville, FL, recorded from 1951 through 1980 (SCS 1985). Germination, defined as radical protrusion > 1 mm, was recorded at 7, 14, and 21 days after sowing. Germination of dark-incubated achenes was recorded in a darkroom under green light (GE 25-W Crystal Color Party! Fiesta! Party Light, Cleveland, OH). After each 21-day germination count during the testing of the 2002 C. leavenworthii, G. pulchella, and R. hirta lots, nongerminated achenes were tested for viability utilizing a 1% 2,3,5-triphenyltetrazolium chloride (TZ) (Sigma-Aldrich, St. Louis, MO), distilled water solution. Achenes were removed from their Petri dishes, placed in individual 30 ml beakers, and covered with the TZ solution. After 24 to 48 hr at 30C in continuous light, achenes were dissected and classified into four categories: fully red and turgid, partially red and turgid, white and turgid, and non-turgid or lacking embryo. All turgid achenes were considered viable due to variability in TZ uptake between treatments. The coolest germination test temperature (5/20C light), however, was used to evaluate monthly lot viability in all analysis using a monthly viability correction factor during statistical

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32 analysis. The 5/20C light treatment was utilized because, on average, its viability deteriorated the least across alternating temperature regimes. Achenes of the same 2002 C. leavenworthii, G. pulchella, and R. hirta lots were also dry stored for 14, 12, and 12 months respectively in the climate-controlled laboratory in open paper bags exposed to florescent lighting. These achenes were tested in an identical manner to those of all 2002 stratified achenes. The last germination treatment for the 2001-harvested C. leavenworthii achene lot was ignored during analysis due to premature harvest of the achene bag from its stratification container by what appeared to be an animal. Exhumation allowed exposure of moistened achenes to sunlight. The bag was reburied and germination testing of the lot was postponed until after all others had been tested to allow for the elimination of possible error introduction during analysis. Statistical Analysis All statistical analyses were performed using SAS (SAS Institute, 2001, Cary, NC). Germination of viable achenes and fraction of dormant viable achenes were arc-sine square root transformed prior to analysis in order to normalize the data and stabilize variances. A three-way ANOVA was performed to evaluate light, month, and temperature effects and their interactions on viability of achenes, germination of viable achenes, and dormant achenes. The Bonferroni adjustment was used to diminish the inflation of type 1 errors caused by the large amount of data. Scanning Electron Micrograph Room temperature and humidity equilibrated intact and bisected achenes were sputter-coated with a gold-palladium alloy through the use of a Denton Vacuum Desk II sputter coater (Moonachie, NJ). Achene surface morphology and seed coat thickness

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33 images were recorded on a field-emission scanning electron microscope (FESEM, Hitachi S-4000, Japan) at the Interdisciplinary Center for Biotechnology Research in the Electron Microscopy Core Laboratory at the University of Florida, Gainesville.

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CHAPTER 3 RESULTS Generally, the main and interactive effects of light, temperature, and months of stratification on germination of viable achenes for all three species were highly significant (Tables 3-1, 3-2, and 3-3), and especially so for C. leavenworthii (Table 3-1). Gaillardia pulchella had all but one of its interactions significant at the 0.05 level when germination of viable achene data was examined (Table 3-2). Rudbeckia hirta had five of its seven interactions significant at the 0.05 level when germination of viable achene data was examined (Table 3-3). Table 3-1. Main and interactive effects of temperature, light, and months of stratification on germination of viable achenes of C. leavenworthii harvested June 19, 2002. Variables Numerator Degrees of Freedom Denominator Degrees of Freedom F Value P Value Temperature 7 528 49.39 < 0.001 Light 1 528 670.30 < 0.001 Months of stratification 10 528 835.29 < 0.001 Temp. x Light 7 528 34.09 < 0.001 Temp. x Months. 70 528 10.26 < 0.001 Light x Months 10 528 50.12 < 0.001 Temp. x Light x Months 70 528 3.94 < 0.001 2001 Coreopsis leavenworthii The mean mass of a C. leavenworthii achene was 0.14 mg. Due to the lack of post-germination treatment TZ testing and lack of dry storage testing, only the percent total germination and speed of germination for total achenes 34

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35 Table 3-2. Main and interactive effects of temperature, light, and months of stratification on germination of viable achenes of G. pulchella harvested August 12, 2002. Variables Numerator Degrees of Freedom Denominator Degrees of Freedom F Value P Value Temperature 7 605 58.06 < 0.001 Light 1 605 10.29 0.0014 Months of stratification 12 605 336.68 < 0.001 Temp. x Light 7 605 4.47 < 0.001 Temp. x Months 84 605 7.82 < 0.001 Light x Months 12 605 3.55 < 0.001 Temp. x Light x Months 84 605 1.24 0.0832 Table 3-3. Main and interactive effects of temperature, light, and months of stratification on germination of viable achenes of R. hirta harvested August 9, 2002. Variables Numerator Degrees of Freedom Denominator Degrees of Freedom F Value P Value Temperature 7 624 2.16 0.0356 Light 1 624 87.91 < 0.001 Months of stratification 12 624 249.55 < 0.001 Temp. x Light 7 624 1.52 0.1570 Temp. x Months 84 624 1.41 0.0131 Light x Months 12 624 7.38 < 0.001 Temp. x Light x Months 84 624 1.23 0.0910 could be determined for the 2001 harvest. The maximum mean germination for most treatments occurred early in January 2002 after 6 months of stratification, except for the 10/20C dark treatment where maximum germination occurred one month later (Fig. A-1, Table 3-4). The maximum mean (+ standard deviation) percent germination for the 16 treatments ranged from 24.5 + 4.6 % to 77.0 + 5.3 % (Table 3-4). The maximum percent germination (77.0 + 5.3 %) occurred in the presence of light at 10/20C after 6 months of

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36 stratification, but it was statistically equivalent to 10 other treatments of which most also occurred after 6 months of stratification (Table 3-4). A tendency for delayed germination (many achenes taking longer than 7 days to germinate) was evident at most temperature regimes in both the light and dark for achenes freshly harvested to those stratified 3 months (Figs. A-2 and A-3). Achene germination speed at the warmer dark temperatures (15/30, 20/25, 20/30, and 23/33C) was difficult to determine for most months of testing due to low germination (Figs. A-3E through A-3H). After 3 or 4 months of stratification (Oct. or Nov.), there was a general increase in percent germination as of day 7 for all treatments. Germination (between day 0 and 7) continued to increase for most treatments up to 6 months of stratification (Jan.), at which point almost all reached maximum germination. After 6 months of stratification, there was a sharp decline until 8 or 9 months of stratification, when germination was low in all treatments (Fig. A-1). There was another increase in germination after 8 or 9 months of stratification for most treatments that peaked at 10 months of stratification (Fig. A-1). Many cooler temperature (5/20, 10/20, and 10/25C) light and dark treatments exhibited increasing delays in germination (from germination occurring by day 7 to occurring by day 14) from 6 to 10 months of stratification (Figs. A-2A through A-2C and A-3A through A-3C). After 10 months (Apr.), there was another sharp decline in germination for all treatments (Fig. A-1). 2002 Coreopsis leavenworthii The percent germination of viable achenes, the percent total germination, the speed of germination for total achenes, and the viability of total achenes were examined for freshly harvested, monthly stratified, and 12 month dry stored achenes from the 2002

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37 Table 3-4. Maximum percent germination (n = 4 + SD) of total 2001-harvested C. leavenworthii achenes with statistically insignificant months for each treatment. Achenes were given 21 days of different alternating temperature regimes under a 12 hr photoperiod or in total darkness. Value in bold is the overall maximum germination. Values in parentheses are the durations of stratification not significantly different from the observed maximum within the same treatment. Months with (*) are not significantly different from overall observed maximum. Light Alternating temperature regime (C) Germination (%) Months of stratification 5/20 75.5 + 1.7 6* (10*) 10/20 77.0 + 5.3 6 (10*) 10/25 70.5 + 4.2 6* (10) 15/25 73.5 + 1.9 6* 15/30 56.0 + 5.0 6 20/25 70.0 + 4.5 6* (7,10) 20/30 68.0 + 2.9 6* (5,10) 23/33 67.5 + 5.9 6* Dark Alternating temperature regime (C) Germination (%) Months of stratification 5/20 73.0 + 5.1 6* (5,10) 10/20 64.0 + 2.2 7 (10) 10/25 66.0 + 3.9 6 (5,10) 15/25 73.5 + 4.2 6* 15/30 24.5 + 4.6 6 (4) 20/25 58.5 + 1.0 6 20/30 58.5 + 2.9 6 23/33 39.0 + 1.3 6 harvest. After 4 months (Oct.) of stratification, germination of viable achenes for all test conditions was nearly 100% (96.2 + 0.7% to 98.4 + 0.1%), except for the 23/33C

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38 dark treatment achenes, which increased from 93.3 + 1.6 to 97.8 + 0.2% one month later (Nov.) (Fig. A-5H). The percent germination of viable achenes remained high (> 87.8 + 1.1%) for the light treatments through the completion of the testing (through 9 months of stratification). The germination percentages for dark treatments remained high (> 87.1 + 0.8%) for four consecutive months (Oct. through Jan.), then declined. The decline in germination for the dark-treated achenes was most pronounced at higher temperature regimes (15/30, 20/25, 20/30, and 23/33C) (Fig. A-5E through Fig. A-5H). The maximum percent of total germination occurred after 4 to 8 months of stratification for all light treatments and after 6 to 7 months of stratification for all dark treatments (Fig. A-4). The maximum percent of total germination for the 16 treatments ranged from 43.5 + 3.0 to 68.5 + 2.1 % (Table 3-5). Maximum germination (68.5 + 2.1 %) occurred at 20/30C light after 6 months of stratification, but was not significantly different from 10 other treatments (Table 3-5). Achenes at 5/20C light and dark exhibited delayed germination over more months of stratification than higher temperature regimes (Fig. A-6A and Fig. A-7A). Germination at 7 days increased across treatments from fresh harvest through 5 to 7 months of stratification. The most rapid germination was observed after 5 or 6 months (Nov. or Dec.) of stratification for dark treatments and after 5, 6, or 7 months (Nov. through Jan.) for light treatments. Light treatments had slower germination over more months of stratification at cooler temperatures, but germination equivalent to or lower germination than 9-month stratified treatments also had greater total germination when compared to dark treatments. After 5 or 6 (Nov. or Dec.) months of stratification there was a decline in total germination, resulting in higher germination at 7 days of light or

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39 dark as temperature increased. Decreases in germination percentages were greater for dark treatments compared to respective light treatments. Treatments with the lowest temperatures (5/20C light and dark) also exhibited a decrease in germination speeds from 6 and 5 months of stratification, respectively, until the end of the experiment (Figs. A-6 and A-7). The percentage of viable achenes was influenced by light treatment (Fig. A-8). For each temperature treatment, those achenes given light generally maintained a higher percentage of viable achenes over those kept in the dark. The light and dark treatments generally had percent viabilities ranging from 50 to 65% and 25 to 50%, respectively. Most treatments exhibited no significant differences between light and dark treatments when given 7 months of stratification. The treatments stored dry (DS) 14 months (from June 2002 to August 2003) at room temperature exhibited low percent germination of viable achene (1.1 + 1.1% to 38.4 + 2.3%). All but one DS treatments germination was equivalent to or lower than freshly harvested treatments when given the same temperature and light (Table 3-6). Achenes DS also exhibited slowed speeds of germination and lower total percent germination compared to the freshly harvested achenes. The majority of DS treatment viability ranged between 50 and 65%, similar to those of the stratified treatments exposed to light (Table 3-7). The greatest overall observed percent viability was 68.7 + 2.6% for the 14-month DS 15/25C dark treatment, but this was statistically equivalent to a few of the stratified treatments. 2002 Gaillardia pulchella The mean mass of a G. pulchella achene was 1.85 mg.

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40 Preliminary testing of G. pulchella achenes harvested in mid-July 2001 revealed high achene viability and germinability. When tested in accordance with AOSA (1994) standards, 89.2 + 6.0% of the total achenes germinated (n = 8). The majority of Table 3-5. Maximum percent germination (n = 4 + SD) of total 2002-harvested C. leavenworthii achenes with statistically insignificant months for each treatment. Achenes were given 21 days of different alternating temperature regimes under a 12 hr photoperiod or in total darkness. Value in bold is the overall maximum germination. Values in parentheses are the durations of stratification not significantly different from the observed maximum within the same treatment. Months with (*) are not significantly different from overall observed maximum. Light Alternating temperature regime (C) Germination (%) Months of stratification 5/20 62.5 + 5.5 7* (1,2,3,4,5,6,9) 10/20 63.5 + 9.5 3* (2,4,5,6*,7,8,9) 10/25 64.5 + 3.9 7* (3,5,6) 15/25 65.0 + 2.4 3* (1,4*,5,7,8) 15/30 64.5 + 5.1 5* (4,7) 20/25 65.0 + 2.1 5* (6) 20/30 68.5 + 2.1 6 (4*) 23/33 59.5 + 3.0 3 (2) Dark Alternating temperature regime (C) Germination (%) Months of stratification 5/20 65.5 + 6.0 6* (5,9) 10/20 47.5 + 1.3 5 (4,7) 10/25 49.5 + 4.5 5 (4,6) 15/25 53.0 + 2.5 6 (5) 15/30 43.5 + 3.0 6 (5) 20/25 54.5 + 5.7 5 20/30 47.0 + 1.7 5 23/33 47.0 + 5.1 5 germination (85.8 + 8.1%) occurred between days 4 and 10. When a concurrent test (n = 3) was run using KNO3, 90.7 + 4.6% of all achenes germinated. However, during the

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41 KNO3 treatment, 28.7 + 15.3% of the achenes germinated at day 4 of treatment. Following two other preliminary germination tests (n = 8 for each) conducted in mid-August and late October of the same year, only 2.0 + 1.9% of the first and 44.5 + 8.4% of the second set of achenes germinated. This represented 72.7 + 51.8% and 82.4 + 4.8% germination of viable achenes, respectively. Table 3-6. Percent germination (n = 4 + SD) of freshly harvested and 14-month dry stored (DS) viable C. leavenworthii achenes harvested in 2002. Achenes were given a 21-day germination treatment at one of eight different temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to either a 12 hr photoperiod or kept in complete darkness. Values in bold are the observed maximum germination for the freshly harvested achenes and for the 14-month dry-stored (DS) treatment. Light Alternating temperature regime (C) Fresh harvest germination (%) DS 14 months germination (%) 5/20 71.9 + 1.2 1.1 + 1.1 10/20 65.4 + 3.1 31.3 + 2.7 10/25 67.3 + 2.4 24.6 + 2.9 15/25 68.8 + 1.2 25.4 + 5.7 15/30 60.7 + 4.1 33.8 + 3.5 20/25 57.0 + 5.1 32.1 + 3.7 20/30 56.5 + 2.9 33.5 + 3.3 23/33 52.3 + 5.3 28.5 + 2.1 Dark Alternating temperature regime (C) Fresh harvest germination (%) DS 14 months germination (%) 5/20 47.0 + 3.7 2.2 + 1.3 10/20 41.5 + 5.4 19.9 + 0.6 10/25 39.3 + 5.2 28.1 + 4.8 15/25 48.7 + 4.3 38.4 + 2.3 15/30 24.9 + 5.1 29.5 + 5.0 20/25 19.3 + 5.5 26.5 + 3.3 20/30 11.0 + 2.6 16.2 + 4.7 23/33 15.3 + 5.2 26.4 + 3.5

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42 The percent germination of viable achenes, the percent germination of total achenes, the percent germination speed of total achenes, and the viability of total achenes were examined for freshly harvested, monthly stratified, and 12 month dry stored achenes from the 2002 harvest. The maximum percent germination of total stratified achenes for each of the 16 treatments occurred within the first 3 months (freshly harvested, one and two months of stratification) of testing. The maximum percent germination ranged from 28.0 + 5.1 to 42.5 + 4.6% depending on treatment (Table 3-8). Maximum germination (42.5 + 4.6%) occurred in the 23/33C light treatment after one month of stratification and was not significantly different from 10 other treatments, the majority of which also occurred after 1 month of stratification (Table 3-8). Germination of buried achenes also occurred prior to 1 month of stratification and was evident upon harvest. Freshly harvested, 1, and 2 month-stratified treatments achieved relatively high germination percentages (approximately 30 to 40%) regardless of temperature and light treatment. This was followed by a sharp drop in percent germination of total achenes (to approximately 4%) at 3 months of stratification. The drop was followed by a sustained low (most not achieving > 14%) for the remainder of testing across all 16 treatments. Freshly harvested G. pulchella achenes yielded viable achene germination from 79.9 + 3.2% to 93.1 + 0.9%. After 1 and 2 months of stratification, the percent germination range narrowed and increased (from 86.6 + 1.8% to 93.1 + 0.8% and from 97.8 + 0.6% to 98.6 + 0.2%, respectively). After 3 months of stratification, germination of viable achenes increased to 100%, decreasing to > 92% after 4 months for all 16 treatments. Simultaneously, however, achene viability dropped to between 1 and 18%

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43 during these months and did not increase for the remainder of testing. There was a rapid decrease in germination of viable achenes for higher temperatures (15/30, 20/25, 20/30, Table 3-7. Percent viability (n = 4 + SD) of freshly harvested and 14-month dry stored (DS) C. leavenworthii achenes harvested in 2002. Achenes were given a 21-day germination treatment at one of eight different temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to either a 12 hr photoperiod or kept in complete darkness. Values in bold are the observed maximum viability for the freshly harvested achenes and for the (DS) 14-month dry-stored treatment. Light Alternating temperature regime (C) Fresh harvest viability (%) DS 14 months viability (%) 5/20 63.0 + 2.8 42.7 + 0.5 10/20 52.0 + 4.0 61.7 + 2.5 10/25 54.7 + 3.6 56.1 + 2.3 15/25 56.7 + 2.3 57.4 + 3.9 15/30 46.2 + 4.5 64.2 + 3.3 20/25 41.4 + 2.3 62.6 + 3.6 20/30 41.0 + 2.6 63.8 + 3.2 23/33 38.2 + 4.0 59.1 + 1.8 Dark Alternating temperature regime (C) Fresh harvest viability (%) DS 14 months viability (%) 5/20 33.8 + 2.7 43.1 + 0.6 10/20 31.0 + 3.3 52.6 + 0.4 10/25 29.6 + 2.3 59.4 + 3.8 15/25 35.0 + 2.7 68.7 + 2.6 15/30 23.7 + 1.5 60.8 + 4.5 20/25 22.1 + 1.5 57.7 + 2.8 20/30 20.0 + 1.2 50.8 + 2.8 23/33 21.0 + 1.2 57.7 + 2.8 and 23/33C) with a more gradual decline for cooler temperatures (5/20, 10/20, 10/25, and 15/25C) after 3 months of stratification. Most treatments reached their lowest

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44 percentage germination of viable achenes between 5 and 7 months of stratification (between January and March). Between 7 and 9 months of stratification, an increase occurred throughout all treatments in the percent germination of viable achenes. This increase culminated when all treatments again reached 100% germination of viable achenes at 11-months of stratification (July). The second occurrence of maximum germination of viable achenes (100%), however, was based on less than 16 + 9 of a possible 100 achenes germinating for any one treatment (Fig. B-1). After having been DS for 12 months, the germination of viable achenes ranged from 98.3 + 0.1% to 98.9 + 0.1% for all 16 treatments. Viable achene germination of DS achenes was almost equivalent to the maximum values set for each stratified treatment and was higher than observed for freshly harvested achenes (Table 3-9). Delayed germination was most evident in freshly harvested achenes (Aug.) (Figs. B-2 and B-3). Freshly harvested low temperature light treatments generally had a slower speed of germination (Figs. B-2A and B-2B). The speed of germination of DS achenes was generally more rapid than freshly harvested achenes and was virtually equivalent to those achenes stratified 1 and 2 months within a given treatment. The percent viability of total achenes followed the same trend as the percent germination of total achenes for all 16 treatments. This occurred because most of the viable achenes germinated each month. During the months when viable achene germination was the lowest (< 50%), the percent of viable achenes from which germination could occur was also greatly reduced. There were only negligible differences between total viability and total germination. The maximum percent viability for each

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45 treatment occurred within the first 3 months of testing and ranged from 28.9 + 2.5% to 46.8 + 4.9%. After 2 months of stratification, there was a decline in percent viability Table 3-8. Maximum percent germination (n = 4 + SD) of total 2002-harvested G. pulchella achenes with statistically insignificant months for each treatment. Achenes were given a 21-day germination treatment at one of eight temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to a 12 hr photoperiod or kept in complete darkness. Value in bold is the overall maximum germination. Values in parentheses are the durations of stratification not significantly different from the observed maximum within the same treatment. Months with (*) are not significantly different from overall observed maximum. Light Alternating temperature regime (C) Germination (%) Months of stratification 5/20 39.0 + 2.6 1* (2*) 10/20 36.0 + 2.2 2* (1) 10/25 37.5 + 3.4 1* 15/25 36.5 + 3.9 1* 15/30 28.0 + 2.7 2 20/25 42.0 + 3.3 1* (2*) 20/30 38.5 + 3.6 1* 23/33 42.5 + 4.6 1 Dark Alternating temperature regime (C) Germination (%) Months of stratification 5/20 38.0 + 2.8 1* 10/20 36.5 + 6.1 0* (1,2) 10/25 32.0 + 3.7 0 (2) 15/25 32.0 + 2.4 1 15/30 30.0 + 3.4 1 (0,2) 20/25 32.0 + 3.7 2 (1) 20/30 30.5 + 1.3 1 23/33 28.0 + 5.1 1 (2)

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46 of total achenes (from a maximum of 18.1 + 1.7% to a minimum of 1.0 + 0.0%) for all 16 treatments, with viability remaining depressed for the remainder of testing (Fig. B-4). The total viability of achenes DS 12 months was almost equivalent to total viability maximums shown by the stratified achenes when compared within each of the 16 treatments (Table 3-10). Achenes DS 12 months (from Aug. 2002 to Aug. 2003) generally exhibited equivalent or higher germination percentages than either achenes freshly harvested or those given any length of stratification (Table 3-11). 2002 Rudbeckia hirta The mean mass of a R. hirta achene was 0.273 mg. Percent germination of viable achenes, percent germination of total achenes, percent germination speed of total achenes, and viability of total achenes were examined for freshly harvested, monthly stratified, and 12 month dry stored achenes from the 2002 harvest. The maximum percent germination of total achenes for 7 of the 16 treatments occurred in the freshly harvested achenes (Fig. C-1). The maximum percent germination of freshly harvested treatments ranged from 52.0 + 2.4% (23/33C light) to 12.0 + 2.2% (20/30C dark). Maximum germination occurred from fresh harvest to the last month of stratification and ranged from 26.0 + 4.8 (15/25C dark stratified 10 months) to 52.0 + Table 3-9. Percent germination of viable (n = 4 + SD) freshly harvested and 12-month dry stored (DS) G. pulchella achenes harvested in 2002. Achenes were given a 21-day germination treatment at one of eight different temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to either a 12 hr photoperiod or kept in complete darkness. Light Alternating temperature regime (C) Fresh harvest germination (%) DS 12 months germination (%) 5/20 90.7 + 1.1 98.7 + 0.2 10/20 90.8 + 0.6 98.6 + 0.2

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47 Table 3-9 Continued Light Alternating temperature regime (C) Fresh harvest germination (%) DS 12 months germination (%) 10/25 90.5 + 1.5 98.6 + 0.2 15/25 90.7 + 1.6 98.7 + 0.2 15/30 79.9 + 3.2 98.5 + 0.1 20/25 90.4 + 1.5 98.7 + 0.2 20/30 80.4 + 3.4 98.8 + 0.1 23/33 91.6 + 0.8 98.8 + 0.1 Dark Alternating temperature regime (C) Fresh harvest germination (%) DS 12 months germination (%) 5/20 92.2 + 0.4 98.8 + 0.2 10/20 93.1 + 0.9 98.6 + 0.1 10/25 92.4 + 1.0 98.4 + 0.3 15/25 91.6 + 0.5 98.4 + 0.4 15/30 91.6 + 1.3 98.5 + 0.1 20/25 89.1 + 1.8 98.8 + 0.0 20/30 89.0 + 0.9 98.3 + 0.1 23/33 88.6 + 0.8 98.6 + 0.1 2.4% (23/33C light freshly harvested) (Table 3-12). The freshly harvested 23/33C light treatment yielding the maximum germination percent (52.0 + 2.4%) was not significantly different from two other freshly harvested treatments (10/20C light and 15/25C light). Germination of buried achenes occurred prior to 4 months of stratification and was evident upon harvest (Fig. 3-1). Percent total germination for all treatments generally

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48 Table 3-10. Percent viability (n = 4 + SD) of freshly harvested and 12-month dry stored (DS) G. pulchella achenes harvested in 2002. Achenes were given a 21-day germination treatment at one of eight different temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to either a 12 hr photoperiod or kept in complete darkness. Values in bold are the observed maximum viability for the freshly harvested achenes and for the 12-month dry-stored (DS) treatment. Light Alternating temperature regime (C) Fresh harvest viability (%) DS 12 months viability (%) 5/20 27.9 + 3.0 39.5 + 4.3 10/20 27.6 + 1.9 37.3 + 5.8 10/25 28.2 + 4.0 38.5 + 4.7 15/25 29.3 + 4.7 40.6 + 6.2 15/30 13.3 + 1.7 34.4 + 3.1 20/25 27.8 + 4.0 40.4 + 4.5 20/30 14.8 + 3.7 42.6 + 2.3 23/33 30.8 + 2.9 41.1 + 3.1 Dark Alternating temperature regime (C) Fresh harvest viability (%) DS 12 months viability (%) 5/20 32.5 + 1.7 44.1 + 5.7 10/20 38.8 + 6.1 35.7 + 2.5 10/25 34.5 + 4.2 34.2 + 6.1 15/25 30.3 + 1.7 34.0 + 5.7 15/30 31.6 + 4.0 33.5 + 3.5 20/25 25.1 + 4.0 43.6 + 1.9 20/30 23.3 + 1.9 29.7 + 2.6 23/33 22.4 + 1.6 36.6 + 3.4 declined after achenes were freshly harvested and reached their lowest after 6 months of stratification (February). Germination then increased until the end of testing (11 months of stratification) (July). Freshly harvested achene germination was usually equivalent or slightly greater than that of achenes stratified 11 months within a treatment. Achenes DS

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49 Table 3-11. Maximum percent germination (n = 4 + SD) of stratified and 12-month dry stored (DS) G. pulchella achenes harvested in 2002. Achenes were given a 21-day germination treatment at one of eight different temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to either a 12 hr photoperiod or kept in complete darkness. Values in bold are the observed maximum germination for the stratified treatments and for the 12-month dry-stored (DS) treatment. Light Alternating temperature regime (C) Germination (%) Months of stratification DS 12 months germination (%) 5/20 39.0 + 2.6 1 39.0 + 3.0 10/20 36.0 + 2.2 2 38.0 + 2.4 10/25 37.5 + 3.4 1 38.5 + 1.7 15/25 36.5 + 3.9 1 40.0 + 3.8 15/30 28.0 + 2.7 2 32.5 + 0.8 20/25 42.0 + 3.3 1 39.0 + 1.5 20/30 38.5 + 3.6 1 42.5 + 1.3 23/33 42.5 + 4.6 1 40.5 + 0.0 Dar k Alternating temperature regime (C) Germination (%) Months of stratification DS 12 months germination (%) 5/20 38.0 + 2.8 1 43.0 + 2.2 10/20 36.5 + 6.1 0 35.0 + 1.7 10/25 32.0 + 3.7 0 33.0 + 1.7 15/25 32.0 + 2.4 1 33.5 + 1.4 15/30 30.0 + 3.4 1 32.5 + 0.8 20/25 32.0 + 3.7 2 39.5 + 2.1 20/30 30.5 + 1.3 1 29.0 + 1.2 23/33 28.0 + 5.1 1 35.5 + 1.5 12 months did not germinate as prolifically as either freshly harvested achenes or achenes stratified 11 months. Freshly harvested R. hirta achenes had nearly every viable achene germinate (91.6

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50 Figure 3-1. Germinated R. hirta achenes harvested after 4 months of stratification in their cloth stratification envelope. The bar in the figure is 3.25 cm. + 1.8% to 98.1 + 0.1 %) and did not again achieve > 90% germination for most treatments until after 8 months of stratification (Apr.) (Fig. C-1). Achenes given an 8, 10, and 11 month stratification achieved > 90% germination under most treatments. Viable germinating achenes dropped to between 6.1 + 6.1% and 54.1 + 3.3% when given 6 months stratification (February) across all temperature and light treatments. Viable achenes given light were usually statistically equivalent to or greater than achenes kept in the dark within each temperature treatment. Achenes DS 12 months had lower germination percentages (52.7 + 6.2% to 74.6 + 2.4%) than freshly harvested or treatments stratified for 7 months or more (Table 3-13). The greatest delay in germination observed for light and dark treatments was at 5/20C and the delay generally decreased as the temperature treatment increased. The most rapid speed of germination occurred during the first and final month of stratification, regardless of treatment. Consistent with total germination, germination speed decreased as stratification progressed toward 6 months of stratification (February) then the speed increased until testing terminated (July) (Figs. C-2 and C-3). Speed of

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51 Table 3-12. Maximum percent germination (n = 4 + SD) of total 2002-harvested R. hirta achenes with statistically insignificant months for each treatment. Achenes were given a 21-day germination treatment at one of eight temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to a 12 hr photoperiod or kept in complete darkness. Value in bold is the overall maximum germination. Values in parentheses are the durations of stratification not significantly different from the observed maximum within the same treatment. Months with (*) are not significantly different from overall observed maximum. Light Alternating temperature regime (C) Germination (%) Months of stratification 5/20 27.5 + 6.2 0 (1,2,9,10,11) 10/20 48.5 + 6.1 0* 10/25 34.5 + 1.3 2 15/25 51.5 + 3.3 0* 15/30 32.0 + 2.9 2 (0) 20/25 33.0 + 2.6 2 (11) 20/30 32.5 + 2.4 2 (1,11) 23/33 52.0 + 2.4 0 Dark Alternating temperature regime (C) Germination (%) Months of stratification 5/20 41.5 + 3.1 0 10/20 37.5 + 2.9 0 10/25 29.5 + 5.4 0 (11) 15/25 26.0 + 4.8 10 (0,11) 15/30 35.5 + 6.4 0 20/25 30.0 + 3.2 0 (11) 20/30 35.0 + 5.4 11 23/33 42.5 + 4.0 0 germination for all treatments was generally slower for DS treatments than for either freshly harvested achenes or those stratified 11 months.

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52 Table 3-13. Percent germination of viable (n = 4 + SD) freshly harvested and 12-month dry stored (DS) R. hirta achenes harvested in 2002 for each treatment. Achenes were given a 21-day germination treatment at one of eight different temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to either a 12 hr photoperiod or kept in complete darkness. Light Alternating temperature regime (C) Fresh harvest germination (%) DS 12 months germination (%) 5/20 95.9 + 0.9 65.6 + 6.8 10/20 97.8 + 0.2 65.0 + 6.6 10/25 96.6 + 0.4 73.1 + 5.3 15/25 98.0 + 0.1 71.6 + 3.2 15/30 96.7 + 0.6 74.5 + 7.4 20/25 96.6 + 0.6 70.9 + 4.2 20/30 95.6 + 0.4 70.9 + 4.0 23/33 98.1 + 0.1 73.6 + 4.8 Dark Alternating temperature regime (C) Fresh harvest germination (%) DS 12 months germination (%) 5/20 97.7 + 0.1 61.6 + 4.0 10/20 97.5 + 0.2 63.1 + 1.0 10/25 96.5 + 0.8 74.6 + 2.4 15/25 95.9 + 0.7 66.6 + 5.7 15/30 97.0 + 0.5 56.4 + 3.9 20/25 96.7 + 0.3 52.7 + 6.2 20/30 91.6 + 1.8 64.9 + 5.5 23/33 97.8 + 0.2 68.0 + 2.2 Also, the percentage viable of total achenes for all treatments generally decreased from fresh harvest to between 5 and 8 months of stratification, then viability increased until testing terminated (Fig. C-4, Table 3-14).

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53 Table 3-14. Percent viable (n = 4 + SD) of freshly harvested and 12-month dry stored (DS) viable R. hirta achenes harvested in 2002 for each treatment. Achenes were given a 21-day germination treatment at one of eight different temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33C). Achenes were also exposed to either a 12 hr photoperiod or kept in complete darkness. Light Alternating temperature regime (C) Fresh harvest viability (%) DS 12 months viability (%) 5/20 27.9 + 5.2 30.4 + 4.9 10/20 47.8 + 5.4 30.2 + 5.8 10/25 31.6 + 3.9 38.6 + 6.1 15/25 51.0 + 3.0 34.5 + 3.5 15/30 33.3 + 4.5 44.2 + 8.3 20/25 32.9 + 5.9 34.9 + 5.6 20/30 23.7 + 2.3 34.9 + 5.8 23/33 53.6 + 2.5 38.9 + 5.7 Dark Alternating temperature regime (C) Fresh harvest viability (%) DS 12 months viability (%) 5/20 45.6 + 2.6 25.7 + 3.4 10/20 41.7 + 2.8 25.7 + 0.7 10/25 33.1 + 6.1 38.1 + 3.2 15/25 27.5 + 4.4 32.1 + 7.5 15/30 36.4 + 5.7 22.2 + 1.8 20/25 31.6 + 2.8 21.1 + 2.8 20/30 13.6 + 2.3 29.0 + 4.3 23/33 46.6 + 3.4 29.9 + 2.0

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CHAPTER 4 DISCUSSION AND CONCLUSIONS Coreopsis leavenworthii If stratified under ambient Florida temperatures, primary physiological conditional dormancy of commercially produced north central Florida C. leavenworthii achenes harvested in mid-June is broken between the end of October and the beginning of January with a type-1 response pattern. The 2002 harvests percent germination of viable stratified achenes ranged from 93.3 + 1.6% to 98.4 + 0.1% during this time over a wide range of temperature regimes in both the light and dark. High total achene germination and an increase in the speed of germination (large proportion of achenes germinating prior to 7 days of treatment) during both years of study also supported this conclusion. The last germination treatment for the 2001-harvested achene lot (6/4/02) was ignored during analysis due to premature harvest of the achene bag from its stratification container, thus allowing exposure of moistened achenes to sunlight. A phytochrome response is hypothesized to have initiated germination upon reburial (Bewley and Black 1994). The depth of burial (7 cm) and limited achene embryo energy stores would have proven a lethal combination if germination were to have occurred prior to proper harvest (Bewley and Black 1994). It could not be assumed that the resulting final months low germination was independent of the premature sunlight exposure; so all treatments from this length of stratification were eliminated from further scrutiny. Most of the more informative aspects of the 2001 and 2002 harvests could not be compared to each other 54

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55 directly because of the lack of TZ testing in the 2001 harvest. The only comparisons that were made were from total germination and speed of germination. During both years of testing, soon after harvest (mid-June) many C. leavenworthii achenes appeared to be conditionally dormant. Dormancy was evident from slow germination speeds (most viable achenes requiring more than 7 days to germinate) during both years and suppressed viable achene germination (ranging from 25 to 89% dormant, depending on treatment) of the 2002 harvest. Freshly harvested achenes were most responsive when germinated in the light at cooler temperatures and they were least responsive when germinated in the dark at warmer temperatures. For the 2002 harvest, achenes broke conditional dormancy after 4 months of stratification for all treatments. During the months leading up to conditional dormancy break, achenes gradually germinated to increasingly higher percentages at warmer temperatures. This indicated that conditional dormancy was fully broken by October exhibiting a type-1 response pattern. From the time achenes became nondormant until the study ended, viable achenes would germinate as long as they were provided with light. Germination of viable achenes decreased after 5 or 6 months of stratification when achenes were not provided light indicating a secondary conditional dormancy; this trend continued until testing ended. This decrease in germination and secondary conditional dormancy onset was hastened in the dark treatments as germination temperature regimes increased. Also, as temperature regimes increased in the dark treatments, there was a reduction in achene viability. Reduced viability may have led to an exaggeration in the decrease observed in viable achene germination, which might have occurred because the basis for determining

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56 the decline in dark-treatment viable-achene germination is intimately associated with total achene viability. Dark achenes appeared to enter a secondary conditional dormancy after approximately 6 or 7 months of stratification. However, the relationship between total lot viability and high temperature dark achene deterioration makes this deduction somewhat speculative. Achenes exposed to light, on the other hand, are believed to have shown a phytochrome response, thus allowing viable achene germination to remain high after conditional dormancy was broken. Not only did light improve germination, it also maintained viability over the dark treatments each month after they became nondormant. This resulted in a larger number of viable achenes from which germination could occur. Darkness also significantly degraded lot viability, thus reducing the germination potential. After conditional dormancy break, viability appeared to be negatively affected by an increase in germination temperature and the absence of light within each month of stratification. As stratification duration lengthened after achenes became nondormant, it, too, negatively affected achene viability. The environmental parameters (moisture requirements, light exposure usually found growing, etc.) of the species and the state of the seed at dispersal (dormancy, viability, light requirements, favorable germination temperature) should be taken into account when deciding on an area to sow seed. As seen from this study, a portion of mid-June harvested C. leavenworthii achenes are conditionally dormant upon dispersal when commercially produced in north central peninsular Florida. The most expedient way for the achenes to break conditional dormancy when dispersed into a natural setting would appear to be 4 to 6 months of stratification (until October to early January if dispersed in

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57 June). In the native habitat a stratification of this type might occur if achenes were buried (entered into the seed bank) to a depth greater than that of sunlight penetration (approximately 2-10 mm depending on the soil particle size, color, and moisture content) (Baskin and Baskin 2001). Then, after 4 to 6 months, the achenes would ideally be brought to the surface to a depth where they could receive exposure to light but still be buried enough so the surrounding soil could provide sufficient moisture to break quiescence. The moist habitats (wet pine flatwoods, glades, prairies, and ruderal wet ditches) in which C. leavenworthii is commonly found should be able to provide the water necessary for germination and sustained growth (Ledin 1951; Taylor 1998). Conditional dormancy should remain broken for at least one year from dispersal and possibly beyond provided light continues to be available to the achenes. During an average December in Gainesville, Florida (the same time C. leavenworthii achenes became nondormant), the mean high and low temperature would typically be between 21 and 8C, respectively (SCS 1985). This cool December temperature is very similar to the 5/20C or 10/20C treatments, which usually retained high viability and also usually had a large percentage of viable achenes germinate as compared to other temperature regimes over the year of stratification. The data supports a late fall to early winter sowing if achenes are nondormant due to a proper pretreatment to break the achenes conditional dormancy. Evidence of successful winter seeding has proven to be more reliable in some species than those performed in the summer despite the lack of dependable moisture (Pfaff et al. 2002). This is believed to occur because of cooler soil temperatures or a reduction in weed competition during this time of year. However, sowing achenes soon after harvest (summer to fall) in a low moist area and sowing deep enough to prevent

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58 light exposure (> 1 cm) may act as an effective pretreatment/stratification. The natural dormancy breaking process may need to be artificially streamlined by timing procedures in order to achieve a uniform C. leavenworthii stand. A consistently moist environment similar to that provided in this thesis may be crucial in overcoming dormancy within 4 to 6 months. A mid-October tilling to the depth of the achenes would bring achenes to the surface, exposing them to light and would remove them from a possible anoxic environment while still retaining a moist environment. This procedure would make an attempt at approximating experimental procedures that effectively overcame C. leavenworthiis primary conditional dormancy (93.3 + 1.6% to 98.4 + 0.1% germination of viable achenes). This however may or may not be practical for FDOT procedures. Without light, viability tends to be lost and the chance of the achenes entering a secondary conditional dormancy increases, especially when temperature increases nearer to 33C. Achenes could experience this situation if they were to remain close enough to the soils surface to experience solar heating, but were buried deep enough to prevent sunlight exposure. This translates into reduced viability and the implementation of a secondary conditional dormancy in achenes that remain buried in the seed bank during moist summer months of the following year (May through Sept.). Achenes probably experience a phytochrome response after 4 months of stratification to delay secondary conditional dormancy onset as long as favorable germination conditions are present. The steadily lengthening of time required for germination to occur, observed at some of the cooler temperature regimes when C. leavenworthii germination speed was examined, would protect achenes from germinating unless there was a significantly prolonged period of favorable germination conditions.

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59 For C. leavenworthii achenes, the effect of dry storage (DS) for approximately one year from harvest remains unresolved. Because of limited testing, it is unclear whether conditional dormancy was broken prior to 14 months of dry storage. It is evident, however, that 14 months of dry storage is an ineffective dormancy-breaking treatment. Germination of dry-stored achenes was negligible (62 to 99% remained dormant) when compared to that of any stratified treatment. In many instances, DS treatments developed a deeper dormancy when compared to that of any stratification length of a similar treatment. The increased dormancy was recognizable from the large number of viable achenes that did not germinate and from slow germination. Dry storage is, however, effective at maintaining achene viability for more than one year. If C. leavenworthii achenes are stored for 14 months then a dormancy breaking treatment will likely need to be employed prior to sowing in order for germination to occur. While C. tinctoria is most closely related phylogenetically to C. leavenworthii and both species occupy wet, open canopy sites, their germination requirements do not seem to have many similarities (Kim et al. 1999; Wunderlin and Hansen 2003). The AOSA (1994) suggests a constant 20C germination environment for C. tinctoria. Kaspar and McWilliams (1982) demonstrated that an illuminated constant 20, 25, or 30C environment yielded favorable germination and achenes did not exhibit dormancy during testing. Conversely, Baskin and Baskin (2001) concluded that an illuminated, alternating 15/25C temperature regime provided optimal germination. However, similar to that of C. leavenworthii, Meyer Elliott (1999) found a mild primary physiological dormancy in C. tinctoria. Meyer Elliott (1999) broke dormancy in C. tinctoria after 3 months of dry storage at 25C and dormancy remained broken for the remainder of testing (7 months).

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60 Coreopsis leavenworthii, however, did not respond to a 14-month dry storage. Primary physiological conditional dormancy in C. leavenworthii was only broken when stratified and it responded favorably to a combination of alternating cool temperature (5/20C) and 12 hr illumination. One foreseeable obstacle that may be encountered during attempted cultivation of C. leavenworthii outside of its native habitat would stem from insufficient moisture required to break quiescence and allow germination. Judging by its natural habitat (wet pine flatwoods, glades, prairies, and ruderal wet ditches), it may be advisable to sow achenes in moist ditches, depressions, and poorly drained areas that have evidence of standing water (Ledin 1951; Taylor 1998). Soil texture should also be examined prior to sowing (Pfaff et al. 2002). Another possible explanation for poor stand establishment would be the sowing of conditionally dormant achenes. Dormancy as a factor influencing desired stand establishment may be overcome by a simulated moist stratification of freshly harvested achenes for 4 to 6 months, but another option may be the implementation of a chemical pretreatment. Some chemicals can take the place of or reduce the need for light and afterripening in Asteraceae are ethylene and, more commonly, gibberellic acid (commonly applied as GA3, GA4, or GA7) (Bewley and Black 1994). Gaillardia pulchella Gaillardia pulchella achenes harvested in late summer (mid-August) appeared to have a very weak primary physiological conditional dormancy; however, they could be considered nondormant (due to a germination of viable achenes of approximately 90% at almost all treatments. Slow germination (many achenes taking 14 to 21 days to

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61 germinate) and a slightly below total germination of viable achenes characterized the mild conditional dormancy. Prior to 1 month of stratification, many achenes buried in the harvested container had germinated. This event and the subsequent removal of germinated achenes prior to germination testing resulted in a reduction of that months total viability of the achenes tested. If these achenes had not germinated in their stratification container and were able to be included in germination testing, a higher percent viability would have been observed and a higher germination of viable achenes may have been observed. After 2 months of stratification, achene viability and the total amount of achenes that germinated declined, but the percentage of viable germinating achenes increased and remained high for one additional month until the fourth month of stratification, when it decreased slightly. From this, it may be assumed that buried achenes germinated in all stratification containers prior to 3 months of stratification (mid-November). Stratification-container germination would explain the abrupt decline in viability and germinability experienced from mid-November until the end of testing. The mild conditional dormancy of the late summer-harvested achenes broke between fresh harvest and 3 months of stratification, but full dormancy break (all treatments exhibiting 100% germination of viable achenes), at 3 months of stratification, was only based on a low number of viable achenes. So, when achenes are stratified, a sharp loss of viability may be expected following dormancy break if emergence is not achieved. A limited number of achenes remain dormant after mild primary conditional dormancy breaks. These achenes appeared to enter a secondary conditional dormancy

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62 when stratification continued. The majority of those achenes go into dormancy during the cooler months of the year (initiating prior to January) and dormancy continues to deepen as the environmental temperature decreases (through winter). This strategy would allow a few of the achenes that have entered the seed bank to germinate (break dormancy) only after a prolonged time (possibly the next season). As is evident from the study, the majority of surviving viable achenes reached their deepest conditional dormancy within 7 months of stratification at the cooler temperatures (5/20, 10/20, and 10/25, plus 15/25C dark). At warmer temperatures (15/25 light, plus 15/30, 20/25, 20/30, and 23/33C) achenes became deeply dormant sooner (between 5 and 6 months of stratification). This separation in time between cool and warm dormancy onset would allow achene germination if exhumation were to occur or if the achenes were not buried excessively deep when temperatures were cool enough to support successful seedling establishment. During the month of January, the temperature in Gainesville, Florida, which averages 7 to 19C, would be cool enough to delay some of the remaining viable achenes dormancy onset until March when the temperature would be warmer (averaging 11 to 24C in Gainesville, Florida) (Fig. 2-1) (SCS 1985). This temporal separation would also cause surviving achenes to become dormant and remain in the seed bank during an unseasonably warm winter. Across all treatments, the deepest dormancy was followed by another break in dormancy (all treatments again exhibiting 100% germination of viable achenes), which occurred after 11 months of stratification (mid-July). Secondary conditional dormancy appeared to be broken during mid-summer when temperatures are at their warmest

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63 (averaging 22 to 33C). The observation of dormancy and its subsequent break was, however, based on a greatly reduced number of viable achenes. The behavior of DS G. pulchella achenes from the late summer (mid-August) harvest remains unresolved due to limited testing. However, after 12 months of dry storage, achenes were nondormant and they had retained a high level of viability. When DS treatments were compared to stratified treatments, the DS treatments performed as well or better than stratified treatments (equivalent or higher germination of viable achenes, total achene germination, and speed of germination). It appears that dry storage (afterripening) of G. pulchella for approximately one year from harvest would be easier and possibly more effective at breaking dormancy, maintaining viability, and maintaining an ease of implementation for roadside establishment than stratification would be. A plant date one year from harvest of the achene lot studied would be in the middle of August the following year. August in Gainesville and throughout the rest of Florida is, however, the hottest time of year (averaging 22 to 33C) (SCS 1985). For those trying to germinate achenes in nonirrigated environments, sowing in August might increase the need for supplemental water. Dry storage durations of greater than or less than a year from harvest may also provide similar results, but this would need to be examined in future studies. Ideally, the achenes would be sown just prior to the season of most dependable moisture (the rainy season usually begins in June) to alleviate the need for externally applied moisture (Pfaff et al. 2002). The months in which the most precipitation occurs, however, are also the warmest months of the year. Freshly-harvested, viable achenes have the ability to germinate in high percentages, so freshly harvested achenes could be sown if time is of utmost importance. This course

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64 of action would require external care for a longer duration (for example, at least two weeks of supplemental irrigation) due to the slow speed of germination found in fresh achenes. A slightly lower proportion of viable achene germination would also be expected. Light does not seem to play a crucial role in the germination process and only negligible differences between light and dark treatments were observed in viable achene germination. This would allow achenes to be sown under a light cover of soil, which would provide for better moisture retention around the achenes. Gaillardia pulchella achenes do not have a large amount of stored energy due to their small size and should most likely not be buried too deeply. Problems currently experienced with stand establishment are most likely related to water requirements of achenes prior to germination and throughout seedling establishment. Sufficient water is critical if dormancy has not been fully broken. Many biotic and abiotic factors can affect viability. As seen from preliminary testing, the viability of different harvests during various months is highly variable. An unsatisfactory stand may result not from the treatment provided, but from poor initial viability. A preliminary TZ test in accordance with AOSA (1994) standards or simply pinching a small sample of imbibed achenes with tweezers in order to find turgid embryos would provide valuable information when determining seeding rate prior to sowing. During preliminary testing, a 2001 mid-July harvest exhibited almost 90% germination. This is compelling evidence, although it does not prove conclusively that July is the ideal time to harvest. A preliminary 2001 mid-August harvest had only 2.8% viability out of the 400 achenes tested, but a mid-August harvest the following year (the 2002 lot studied in detail) had a fresh harvest viability average of 27.4% when 3200

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65 achenes were put through variable temperature regime germination testing prior to viability testing. Evidently, there may be a great deal of viability variation among months within a year and between successive years, so viability testing is critical before the purchase of any lot of achenes. Preliminary testing also hinted that although a large number of achenes may not appear to have germinated, the amount of germinating viable achenes may have been substantially high. A viable achene germination of 72.7% (mid-August), 82.4% (late October), > 89.2% (mid-July, value taken from germination of total not germination of viable achenes), and 89.5% (mean from the fresh achene viability of the 2002 lot studied in detail) seems to demonstrate that, regardless of lot viability, a large proportion of freshly harvested viable G. pulchella achenes can be expected to germinate when favorable conditions are present. Rudbeckia hirta Most R. hirta achenes appear non-dormant upon fresh harvest in late summer (mid-August). This is evident from high viable achene germination (> 95%) in all but one treatment (20/30C dark). Most freshly harvested achenes initiated germination prior to 7 days of treatment in all but the coolest treatments (5/20C light and dark treatments). The majority of germination in both 5/20C treatments occurred between 7 and 14 days. Low temperatures are believed to significantly affect the physiological mechanisms responsible for germination through slowed enzyme reactions resulting in sluggish germination (Bewley and Black 1994). As previously stated, the literature recommends sowing R. hirta achenes in the late fall or early winter (Sept. through Dec.) so achenes may be produced by the following summer (Norcini and Aldrich 2003, Norcini et al.

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66 1999, Pfaff et al. 2002). Over the following 6 months of stratification, achenes on average gradually entered a physiological conditional dormancy. The progression into dormancy was characterized by lowered germination of viable achenes and lowered germination of total achenes and from a slowed speed of achene germination. After 4 months of stratification, though, achene germination prior to harvest occurred in the stratification container. As seen for G. pulchella, this event is believed to have reduced the initial viability and observed germination due to the removal of pre-germinated achenes prior to germination testing. Pre-germinated achenes were subsequently not factored into the fourth months germination totals. This removal of a number of non-dormant, viable achenes may have caused the depression in germination of viable achenes for that month. The only significant dormancy incurred was at 6 months of stratification (February) when the germination of viable achenes ranged from 6.1 + 6.1 to 54.1 + 3.3%. Conditional dormancy broke 5 months later (July) and was characterized by an increased germination speed (achene germination prior to 7 days of treatment), greater viable achene germination (from 88.9 + 3.3 to 93.4 + 0.5%), and greater total achene germination during all test treatments. The erratic nature of the R. hirta germination of viable achene results may stem from the way in which this value is calculated. This point can best be demonstrated by an exaggerated example. When the only viable achene germinates during a germination test 100% of viable achenes germinated. The previous situation appears equivalent to a germination test where 200 viable achenes germinated which would also appear as 100% germination of viable achenes. So, with a low initial viability a difference of just a few

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67 germinating or dormant viable achenes can affect the calculated percentage of germinating viable achenes greatly. The lack of pregermination testing of viability may have also affected the calculations due to possible viability determination errors. This is discussed further in Triphenyltetrazolium Chloride Testing Technique section at the end of this chapter. The behavior of DS R. hirta achenes from a late summer (mid-August) harvest to approximately one year later remains unresolved. It could not be determined if conditional dormancy became deeper or if it was fully overcome during the prior 11 months of storage due to limited testing. After 12 months of dry storage, though, all achenes exposed to the various germination treatments showed signs of having developed a deeper dormancy over freshly harvested or 11-month stratified achenes. For most treatments, this was evident from lower germination of viable achenes and was reinforced by essentially the same viability as freshly harvested or 11-month stratified achenes. Dry storage evidently is not an effective dormancy breaking treatment, but it can maintain viability. Across all treatments, there was an unexpected, gradual dip and subsequent rise in total viability during the course of testing. One possible explanation for this phenomenon is that dormant achenes exposed to the rigors of the germination process have a hampered ability to retain viability as compared to achenes that delay germination until they are in a state of non-dormancy. The need for pre-treatment viability (TZ) testing is important in any future study of this species because achene viability appears to be greatly affected by germination testing. Bnyai and Barabs (2002) state that viability and expected

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68 germination should be equivalent as long as dormancy is not present and as long as a seed lot has not been deteriorated from a germination test of normal or extended duration. Triphenyltetrazolium Chloride Testing Technique There should be no significant difference among the total viability of subsamples of a particular seed lot when total percent viable seed is calculated as percent germinated seed plus dormant seed (based on a post germination TZ test) as long as the germination test itself does not reduce viability. However, this was not the case in our work, and highlights the need for additional research about TZ testing standards. The coolest germination test temperature (5/20C light) was used to evaluate monthly viability during statistical analysis through the employment of a monthly viability correction factor. The 5/20C light treatment was used because the viability of achenes in this germination test regime deteriorated the least on average across monthly stratification treatments. Because each of the three species tested had some degree of dormancy and because of reduced monthly lot viability associated with an increase in germination treatment temperatures, viability was assigned to turgid achenes instead of those testing positive in the post-germination TZ tests in order to reduce false negatives (Baskin and Baskin 2001; J.G. Norcini personal communication). The determination of viability from turgid embryos after a germination test, however, still does not appear to be able to determine initial pregermination testing viability. Because seeds have been known to give faulty TZ results if dormant and/or deteriorated from a germination test, it would be advisable in future studies involving these three species to perform a pre-germination TZ test to each stratified lot upon harvest and post-germination TZ tests on all nongerminated achenes. This procedure should help to minimize and quantify the effect of germination test

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69 deterioration. It would also provide more insight into the effect that temperature and light treatments had on viability. It is also advisable to track turgid embryos in order to eliminate the possibility of TZ testing error as a result of a previously unknown dormancy in an unstudied species. In a study of this nature, dormancy may be anticipated, but not avoided, as a possible factor influencing TZ testing. Implications for Roadside Plantings Coreopsis leavenworthii (mid-June Harvest) 55 to 70% of freshly harvested viable achenes may be able to germinate if sown close enough to the soil surface to receive light and provided sufficient moisture is available Sow shallowly (< 7 mm), due to limited energy storage, and for needed moisture retention (Baskin and Baskin 2001, Norcini et al. 1999) Sow in low areas due to species moist habitat background and to reduce the need for supplemental water Dormancy may be lost 4 to 6 months after sowing if dark stratified at equivalent Gainesville, Florida temperatures If dark stratification continues and germination does not occur, achenes tend to enter a secondary dormancy In drier sites, water thoroughly for at least 2 weeks to facilitate germination Mow in the late fall after achene development to disperse mature achenes A 14-month dry storage for can maintain viability

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70 Gaillardia pulchella (Late Season Harvest) Achenes stored dry in a climate controlled facility for 1 year will germinate more rapidly and uniformly than freshly harvested achenes Mild primary dormancy is broken prior to 1 month when stratified A few achenes (< 10%) can be expected to enter a secondary dormancy if stratified Sow shallowly (< 1 cm) due to limited energy storage and for needed moisture retention (Baskin and Baskin 2001, Norcini et al. 1999) Sow in the early fall If sowing one year dry stored achenes, keep soil moist for at least 1 week If sowing freshly harvested achenes, keep soil moist for at least 2 weeks due to slowed germination of approximately half the achenes able to germinate Mow in the late fall after achene development to disperse mature achenes A 12-month dry storage can maintain viability Rudbeckia hirta (Late Season Harvest) Physiological dormancy seems to develop soon after maturation when achenes are stratified Dormancy is deepest 6 months after maturation when stratified and can be expected to take one year to become nondormant Sow freshly harvested achenes shallowly (< 7 mm) due to limited energy storage, needed moisture retention, and to provide light to the achene (Baskin and Baskin 2001, Norcini et al. 1999) Sow in the fall

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71 Sow freshly harvested achenes and keep soil moist for at least 2 weeks due to slowed germination of up to half the achenes able to germinate Mow in the late fall after achene development to disperse mature achenes A 12-month dry storage only degrades viability minimally

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APPENDIX A Coreopsis leavenworthii FIGURES 01020304050607080907/48/39/210/211/112/112/311/303/13/314/305/30Day germination treatment initiated (30 day scale)Germination (%) 5/20C Light 5/20C DarkA Figure A-1. Mean (n = 4) percent germination (+ SD) of total 2001-harvested C. leavenworthii achenes. Achenes were subjected to a 21-day germination treatment for both a 12 hr photoperiod and in complete darkness at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification. 72

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73 01020304050607080907/48/39/210/211/112/112/311/303/13/314/305/30Day germination treatment initiated (30 day scale)Germination (%) 10/20C Light 10/20C DarkB 01020304050607080907/48/39/210/211/112/112/311/303/13/314/305/30Day germination treatment initiated (30 day scale)Germination (%) 10/25C Light 10/25C DarkC Figure A-1. Continued

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74 01020304050607080907/48/39/210/211/112/112/311/303/13/314/305/30Day germination treatment initiated (30 day scale)Germination (%) 15/25C Light 15/25C DarkD 01020304050607080907/48/39/210/211/112/112/311/303/13/314/305/30Day germination treatment initiated (30 day scale)Germination (%) 15/30C Light 15/30C DarkE Figure A-1. Continued

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75 01020304050607080907/48/39/210/211/112/112/311/303/13/314/305/30Day germination treatment initiated (30 day scale)Germination (%) 20/25C Light 20/25C DarkF 01020304050607080907/48/39/210/211/112/112/311/303/13/314/305/30Day germination treatment initiated (30 day scale)Germination (%) 20/30C Light 20/30C DarkG Figure A-1. Continued

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76 01020304050607080907/48/39/210/211/112/112/311/303/13/314/305/30Day germination treatment initiated (30 day scale)Germination (%) 23/33C Light 23/33C DarkH Figure A-1. Continued 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day A Figure A-2. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2001-harvested C. leavenworthii achenes at a 12 hr photoperiod. Achenes were subjected to a 21-day germination treatment, at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification.

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77 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day B 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day C Figure A-2. Continued

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78 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day D 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day E Figure A-2. Continued

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79 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day F 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day G Figure A-2. Continued

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80 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day H Figure A-2. Continued 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day A Figure A-3. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2001-harvested C. leavenworthii achenes in complete darkness. Achenes were subjected to a 21-day germination treatment, at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification.

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81 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day B 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day C Figure A-3. Continued

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82 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day D 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day E Figure A-3. Continued

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83 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day F 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day G Figure A-3. Continued

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84 0102030405060708090J A S O N D J F M A M J MonthGermination (%) 7 Day 14 Day 21 Day H Figure A-3. Continued 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 5/20C Light 5/20C DarkA Figure A-4. Mean (n = 4) percent germination (+ SD) of total 2002-harvested C. leavenworthii achenes. Achenes were subjected to a 21-day germination treatment for both a 12 hr photoperiod and in complete darkness at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 9 months of stratification.

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85 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 10/20C Light 10/20C DarkB 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 10/25C Light 10/25C DarkC Figure A-4. Continued

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86 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 15/25C Light 15/25C DarkD 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 15/30C Light 15/30C DarkE Figure A-4. Continued

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87 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 20/25C Light 20/25C DarkF 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 20/30C Light 20/30C DarkG Figure A-4. Continued

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88 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 23/33C Light 23/33C DarkH Figure A-4. Continued 01020304050607080901006/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 5/20C Light 5/20C DarkA Figure A-5. Mean (n = 4) percent germination (+ SD) of viable 2002-harvested C. leavenworthii achenes. Achenes were subjected to a 21-day germination treatment for both a 12 hr photoperiod and in complete darkness at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 9 months of stratification.

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89 01020304050607080901006/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 10/20C Light 10/20C DarkB 01020304050607080901006/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 10/25C Light 10/25C DarkC Figure A-5. Continued

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90 01020304050607080901006/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 15/25C Light 15/25C DarkD 01020304050607080901006/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 15/30C Light 15/30C DarkE Figure A-5. Continued

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91 01020304050607080901006/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 20/25C Light 20/25C DarkF 01020304050607080901006/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 20/30C Light 20/30C DarkG Figure A-5. Continued

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92 01020304050607080901006/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Germination (%) 23/33C Light 23/33C DarkH Figure A-5. Continued 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayA Figure A-6. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested C. leavenworthii achenes at a 12 hr photoperiod. Achenes were subjected to a 21-day germination treatment at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 9 months of stratification and achenes exposed to the previous conditions, but dry-stored (DS) 14 months at room temperature.

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93 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayB 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayC Figure A-6. Continued

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94 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayD 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayE Figure A-6. Continued

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95 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayF 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayG Figure A-6. Continued

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96 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayH Figure A-6. Continued 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayA Figure A-7. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested C. leavenworthii achenes in complete darkness. Achenes were subjected to a 21-day germination treatment at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 9 months of stratification and achenes exposed to the previous conditions, but dry-stored (DS) 14 months at room temperature.

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97 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayB 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayC Figure A-7. Continued

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98 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayD 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayE Figure A-7. Continued

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99 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayF 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayG Figure A-7. Continued

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100 010203040506070J J A S O N D J F M AugDSMonthGermination (%) 7 Day 14 Day 21 DayH Figure A-7. Continued 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Viable (%) 5/20C Light 5/20C DarkA Figure A-8. Mean (n = 4) percent viability (+ SD) of 2002-harvested C. leavenworthii achenes. Achenes were subjected to a 21-day germination treatment for a 12 hr photoperiod and in complete darkness at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 9 months of stratification.

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101 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Viable (%) 10/20C Light 10/20C DarkB 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Viable (%) 10/25C Light 10/25C DarkC Figure A-8. Continued

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102 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Viable (%) 15/25C Light 15/25C DarkD 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Viable (%) 15/30C Light 15/30C DarkE Figure A-8. Continued

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103 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Viable (%) 20/25C Light 20/25C DarkF 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Viable (%) 20/30C Light 20/30C DarkG Figure A-8. Continued

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104 010203040506070806/247/248/239/2210/2211/2112/211/202/193/21Day germination treatment initiated (30 day scale)Viable (%) 23/33C Light 23/33C DarkH Figure A-8. Continued

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APPENDIX B Gaillardia pulchella FIGURES 01020304050607080901001108/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Germination (%) 5/20C Light 5/20C Dark A Figure B-1. Mean (n = 4) percent germination (+ SD) of viable 2002-harvested G. pulchella achenes. Achenes were subjected to a 21-day germination treatment for both a 12 hr photoperiod and in complete darkness at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification. Vertical line represents the date a large number of germinated achenes were observed at harvest. 105

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106 01020304050607080901001108/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Germination (%) 10/20C Light 10/20C Dark B 01020304050607080901001108/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Germination (%) 10/25C Light 10/25C Dark C Figure B-1. Continued

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107 01020304050607080901001108/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Germination (%) 15/25C Light 15/25C Dark D 01020304050607080901001108/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Germination (%) 15/30C Light 15/30C Dark E Figure B-1. Continued

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108 01020304050607080901001108/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Germination (%) 20/25C Light 20/25C Dark F 01020304050607080901001108/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Germination (%) 20/30C Light 20/30C Dark G Figure B-1. Continued

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109 01020304050607080901001108/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Germination (%) 23/33C Light 23/33C Dark H Figure B-1. Continued 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day A Figure B-2. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested G. pulchella achenes at a 12 hr photoperiod. Achenes were subjected to a 21-day germination treatment at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification and achenes exposed to the previous conditions, but dry-stored (DS) 12 months at room temperature. Vertical line represents the date a large number of germinated achenes were observed at harvest

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110 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day B 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day C Figure B-2. Continued

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111 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day D 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day E Figure B-2. Continued

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112 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day F 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day G Figure B-2. Continued

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113 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day H Figure B-2. Continued 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day A Figure B-3. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested G. pulchella achenes in complete darkness. Achenes subjected to a 21-day germination treatment at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification and achenes exposed to the previous conditions, but dry-stored (DS) 12 months at room temperature. Vertical line represents the date a large number of germinated achenes were observed at harvest.

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114 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day B 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day C Figure B-3. Continued

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115 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day D 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day E Figure B-3. Continued

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116 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day F 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day G Figure B-3. Continued

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117 01020304050A S O N D J F M A M J J A -DSMonthGermination ( % 7 Day 14 Day 21 Day H Figure B-3. Continued 05101520253035404550558/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Viable (%) 5/20C Light 5/20C Dark A Figure B-4. Mean (n = 4) percent viability (+ SD) of 2002-harvested G. pulchella achenes. Achenes were subjected to a 21-day germination treatment for both a 12 hr photoperiod and in complete darkness at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification. Vertical line represents the date a large number of germinated achenes were observed at harvest.

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118 05101520253035404550558/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Viable (%) 10/20C Light 10/20C Dark B 05101520253035404550558/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Viable (%) 10/25C Light 10/25C Dark C Figure B-4. Continued

PAGE 132

119 05101520253035404550558/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Viable (%) 15/25C Light 15/25C Dark D 05101520253035404550558/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Viable (%) 15/30C Light 15/30C Dark E Figure B-4. Continued

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120 05101520253035404550558/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Viable (%) 20/25C Light 20/25C Dark F 05101520253035404550558/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Viable (%) 20/30C Light 20/30C Dark G Figure B-4. Continued

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121 05101520253035404550558/209/1910/1911/1812/181/172/163/184/175/176/167/16Day germination treatment initiated (30 day scale)Viable (%) 23/33C Light 23/33C Dark H Figure B-4. Continued

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APPENDIX C Rudbeckia hirta FIGURES 01020304050607080901001108/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Germination (%) 5/20C Light 5/20C Dark A Figure C-1. Mean (n = 4) percent germination (+ SD) of viable 2002-harvested R. hirta achenes. Achenes were subjected to a 21-day germination treatment for both a 12 hr photoperiod and in complete darkness, at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification. Vertical line represents the date of a large number of germinated achenes observed at harvest. 122

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123 01020304050607080901001108/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Germination (%) 10/20C Light 10/20C Dark B 01020304050607080901001108/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Germination (%) 10/25C Light 10/25C Dark C Figure C-1. Continued

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124 01020304050607080901001108/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Germination (%) 15/25C Light 15/25C Dark D 01020304050607080901001108/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Germination (%) 15/30C Light 15/30C Dark E Figure C-1. Continued

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125 01020304050607080901001108/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Germination (%) 20/25C Light 20/25C Dark F 01020304050607080901001108/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Germination (%) 20/30C Light 20/30C Dark G Figure C-1. Continued

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126 01020304050607080901001108/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Germination (%) 23/33C Light 23/33C Dark H Figure C-1. Continued 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day A Figure C-2. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested R. hirta achenes at a 12 hr photoperiod. Achenes were subjected to a 21-day germination treatment at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification and achenes exposed to the previous conditions, but dry-stored (DS) 12 months at room temperature. Vertical line represents the date a large number of germinated achenes were observed at harvest.

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127 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day B 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day C Figure C-2. Continued

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128 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day D 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day E Figure C-2. Continued

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129 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day F 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day G Figure C-2. Continued

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130 01020304050A S O N D J F M A M J J A -DSMonthGermination ( % 7 Day 14 Day 21 Day H Figure C-2. Continued 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day A Figure C-3. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-harvested R. hirta achenes in complete darkness. Achenes subjected to a 21-day germination treatment at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification and achenes exposed to the previous conditions, but dry-stored (DS) 12 months at room temperature. Vertical line represents the date a large number of germinated achenes were observed at harvest.

PAGE 144

131 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day B 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day C Figure C-3. Continued

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132 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day D 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day E Figure C-3. Continued

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133 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day F 01020304050A S O N D J F M A M J J A -DSMonthGermination (%) 7 Day 14 Day 21 Day G Figure C-3. Continued

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134 01020304050A S O N D J F M A M J J A -DSMonthGermination ( % 7 Day 14 Day 21 Day H Figure C-3. Continued 01020304050608/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Viable (%) 5/20C Light 5/20C Dark A Figure C-4. Mean (n = 4) percent viability (+ SD) of total 2002-harvested R. hirta achenes. Achenes were subjected to a 21-day germination treatment for both a 12 hr photoperiod and in complete darkness at each of eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30, and H. 23/33C) over 11 months of stratification. Vertical line represents the date a large number of germinated achenes were observed at harvest.

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135 01020304050608/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Viable (%) 10/20C Light 10/20C Dark B 01020304050608/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Viable (%) 10/25C Light 10/25C Dark C Figure C-4. Continued

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136 01020304050608/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Viable (%) 15/25C Light 15/25C Dark D 01020304050608/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Viable (%) 15/30C Light 15/30C Dark E Figure C-4. Continued

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137 01020304050608/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Viable (%) 20/25C Light 20/25C Dark F 01020304050608/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Viable (%) 20/30C Light 20/30C Dark G Figure C-4. Continued

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138 01020304050608/219/2010/2011/1912/191/182/173/194/185/186/177/17Day germination treatment initiated (30 days)Viable (%) 23/33C Light 23/33C Dark H Figure C-4. Continued

PAGE 152

REFERENCES Association of Official Seed Analysts (AOSA). 1994. Rules for testing seeds. J. Seed Technol. 16(3): 1-113. Atwater, B.R. 1980. Germination, dormancy and morphology of the seeds of herbaceous ornamental plants. Seed Sci. Technol. 8: 523-573. Banovetz, S.J. and S.M. Scheiner. 1994a. Secondary seed dormancy in Coreopsis lanceolata. Amer. Midl. Naturalist 131: 75-83. ----and -----. 1994b. The effects of seed mass on the seed ecology of Coreopsis lanceolata. Amer. Midl. Naturalist 131: 65-74. Bnyai, J. and J. Barabs. 2002. Handbook on statistics in seed testing. Association of Official Seed Analysts. Baskin, C.C., J.M. Baskin, and O.W. Van Auken. 1992. Germination response patterns to temperature during afterripening of achenes of four Texas winter annual Asteraceae. Canad. J. Bot. 70: 2354-2358. ----and -----. 2001. Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. Academic Press, Boston, MA. Bell, R.C. and B.J. Taylor. 1982. Florida Wild Flowers and Roadside Plants. Laurel Hill Press, Chapel Hill, NC. Bewley, J.D. and M. Black. 1994. Physiology of Development and Germination (2nd Ed.). Plenum Press, New York, NY. Capon, B. and W. Van Asdall. 1967. Heat pre-treatment as a means of increasing germination of desert annual seeds. Ecol. 48: 305-306. Carpenter, W.J. and E.R. Ostmark. 1992. Growth regulators and storage temperature govern germination of Coreopsis seed. HortScience 27(11): 1190-1193. Chapman, A.W. 1887. Flora of the Southern United States: Containing an Abridged Description of the Flowering Plants and Ferns of Tennessee, North and South Carolina, Georgia, Alabama, Mississippi, and Florida: Arranged According to the Natural System. Ivison, Blakeman, Taylor, and Co., New York, NY. 139

PAGE 153

140 Crawford, D.J., E.B. Smith, and R.E. Pilatowski. 1984. Isozymes of Coreopsis section Calliopsis (Compositae): genetic variation within and divergence among the species. Brittonia 36(4): 375-381. Fay, A.M., M.A. Bennett, and S.M. Still. 1994. Osmotic seed priming of Rudbeckia fulgida improves germination and expands germination range. HortScience 29(8): 868-870. Florida Department of Transportation (FDOT), Environmental Management Office. 2003. Establishment of Sustainable Populations of Native Wildflowers on Floridas RoadsidesEcosystem Management Approaches to the Statewide Application of FDOTs Roadside Wildflower Program. Proposal for Use of Ecosystem Management Funds. Harkess, R.L. and R.E. Lyons. 1994. Rudbeckia hirta L.: A versatile North American wildflower. HortScience 29(3): 134 and 227. Harper-Lore, B. and M. Wilson. 2000. Roadside Use of Native Plants. Island Press, Washington, D.C. Heywood, J.S. 1993. Biparental inbreeding depression in the self-incompatible annual plant Gaillardia pulchella (Asteraceae). Amer. J. Bot. 80(5): 545-550. Jansen, R.K., E.B. Smith, and D.J. Crawford. 1987. A cladistic study of North American Coreopsis (Asteraceae: Heliantheae). Pl. Syst. Evol. 157: 73-84. Kaspar, M.J. and E.L. McWilliams. 1982. Effects of temperature on the germination of selected wildflower seeds. HortScience 17(4): 595-596. Kim, S.-C., D.J. Crawford, M. Tadesse, M. Berbee, F.R. Ganders, M. Pirseyedi, and E.J. Esselman. 1999. ITS sequences and phylogenetic relationships in Bidens and Coreopsis (Asteraceae). Syst. Bot. 24(3): 480-493. Ledin B.R. 1951. The Compositae of South Florida. J. Florida Acad. Sci.: 151-152. Marois, J.J. and J.G. Norcini. 2003. Survival of black-eyed susan from different regional seed sources under low and high input systems. HortTechnology 13(1): 161-165. Meyer Elliott, S.A. 1999. Coreopsis tinctoria: Germination requirements and competitive ability with a perennial grass. MS thesis. Univ. Tex. San Ant., Tex. Myers, R.L. and J. J. Ewel, eds. 1990. Ecosystems of Florida. University of Central Florida Press, Orlando, FL. Neigebauer, A.L., G.L. Horst, and D.H. Steinegger. 2000. Shoot and root characterization of Rudbeckia hirta L. mowed at different heights. HortScience 35(7): 1247-1248.

PAGE 154

141 Niering, W.A. and N.C. Olmstead. 2001. National Audubon Society Field Guide to North American Wildflowers Eastern Region. Chanticleer Press, Inc., New York, NY. Norcini, J.G., J.H. Aldrich. 2003. Wildflower sowing date demonstration spring update. North Florida Res. Educ. Center Newslett. 5(10): 2-3. -----, -----, L.A. Halsey, and J.G. Lilly. 1998. Seed source affects performance of six wildflower species. Proc. Florida State Hort. Soc. 111: 4-9. -----, M. Thetford, K.A. Klock-Moore, M.L. Bell, B.K. Harbaugh, and J.H. Aldrich. 2001. Growth, flowering, and survival of black-eyed susan from different regional seed sources. HortTechnology 11(1): 26-30. -----, D.J. Zimet, C. Maura, S. Pfaff, and M.A. Gonter. 1999. Seed production of a Florida ecotype of black-eyed susan. Univ. Florida Extens. Inst. Food Agric. Sci. Circ. 1226. Pandey, A.K. and R.P. Singh. 1982. Development and structure of seeds and fruits in composite: Coreopsis species. J. Indian Bot. Soc. 61: 417-425. Pfaff, S., M.A. Gonter, and C. Maura, Jr. 2002. Florida Native Seed Production Manual. USDA-NRCS Brooksville Plant Materials Center and Florida Institute of Phosphate Research, Brooksville, FL. Samfield, D.M., J.M. Zajicek, and B.G. Cobb. 1990. Germination of Coreopsis lanceolata and Echinacea purpurea seeds following priming and storage. HortScience 25(12): 1605-1606. -----, -----, and -----. 1991. Rate and uniformity of herbaceous perennial seed germination and emergence as affected by priming. J. Amer. Soc. Hort. Sci. 116(1): 10-13. SAS Institute. 2001. Windows Version 8.0, SAS Institute, Inc., Cary, NC. Sherff, E.E. 1936. Revision of the genus Coreopsis. Field Mus. Nat. Hist., Bot. Ser. 11 (6), Pub. 366: 430-431. Shipley, B. and M. Parent. 1991. Germination responses of 64 wetland species in relation to seed size, minimum time to reproduction and seedling relative growth rate. Funct. Ecol. 5: 111-118. Smith, E.B. 1978. Notes on Coreopsis. Sida 7: 304-307. -----. 1982. Phyletic trends in section Coreopsis of the genus Coreopsis (Compositae). Bot. Gaz. 143(1): 121-124.

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142 Snow, A.A., P. Moran-Palma, L.H. Rieseberg, A. Wszelaki, and G.J. Seiler. 1998. Fecundity, phenology, and seed dormancy of F1 wild-crop hybrids in sunflower (Helianthus annuus, Asteraceae). Amer. J. Bot. 85(6): 794-801. Soil Conservation Service (USDA). 1985. Soil survey of Alachua County, Florida. Gainesville, FL. Taylor, W.K. 1998. Florida Wildflowers in Their Natural Communities. University Press of Florida, Gainesville, FL. Turner, B.L. and M. Whalen. 1975. Taxonomic study of Gaillardia pulchella (AsteraceaeHeliantheae). Wrightia 5(6): 189-192. Voigt, J.W. 1977. Seed germination of true prairie forbs. J. Range Managem. 30(6): 439-441. Wunderlin, R.P. and B.F. Hansen. 2003. Guide to the Vascular Plants of Florida (2nd Ed.). University Press of Florida, Gainesville, FL.

PAGE 156

BIOGRAPHICAL SKETCH Steven Matthew Kabat was born on September 28, 1976, in Tampa, Florida, to Richard and Joan Kabat. Steven graduated from Bloomingdale Senior High School in 1995 and started his undergraduate degree at the University of South Florida, Tampa. While pursuing his degree, he became interested in the plasticity of plants and was fascinated by various propagation methods shortly before taking his first botany course. His vascular plant course, under the direction of the enthusiastic Dr. Richard Mansell, changed his life in many ways. This is where he learned of the tremendous diversity and complexity of the plants around him and where he first met his future wife. Steven was married to Cathleen Ann Touchton in July of 2000. After earning his Bachelor of Science degree from the University of South Florida in August of 2000, he and his wife began their graduate careers at the University of Florida, Gainesville. Steven was interested in environmental horticulture and all aspects of plant culture and chose to pursue his graduate degree under the guidance of Dr. Bijan Dehgan, while his wife pursued her graduate degree in botany under Dr. Walter Judd. Steven then earned his Master of Science in August of 2004. 143


Permanent Link: http://ufdc.ufl.edu/UFE0006980/00001

Material Information

Title: An Ecologically based study of germination requirements and dormancies in three commercially produced Florida native wildflowers
Physical Description: Mixed Material
Language: English
Creator: Kabat, Steven Matthew ( Dissertant )
Dehgan, Bijan ( Thesis advisor )
Norcini, Jeffrey ( Reviewer )
Gordon, Doria ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2004
Copyright Date: 2004

Subjects

Subjects / Keywords: Horticultural Sciences thesis, M.S.
Dissertations, Academic -- UF -- Horticulture
Spatial Coverage: United States--Florida

Notes

Abstract: Three Florida Asteraceae species were examined for one year to determine germination parameters and presence of dormancy. Coreopsis leavenworthii, Gaillardia pulchella, and Rudbeckia hirta achenes were stratified under ambient Florida temperatures or stored dry at room temperature for one year. Achenes were germinated each month at eight different fluctuating temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33⁰C) in the light or dark. Germination was recorded at 7, 14, and 21 days, and germinated achenes removed. Nongerminated achenes were tested for viability using 2,3,5-triphenyltetrazolium chloride. Both the June harvested C. leavenworthii and the August harvested G. pulchella achenes exhibited a physiological dormancy followed by a secondary dormancy. Late season harvested R. hirta achenes were nondormant and this was followed by a secondary physiological dormancy. Coreopsis leavenworthii's primary physiological dormancy was broken after 4 to 6 months of stratification, which resulted in more than or equal to 96% germination of viable achenes in all but one (23/33⁰C dark) treatment. For the late season harvested G. pulchella, the mild primary physiological dormancy was broken after one month of stratification, which resulted in more than or equal to 87% germination of viable achenes. After dormancy broke, most G. pulchella achenes became inviable. The achenes that remained viable entered a secondary dormancy (approximately less than or equal to 15%). After one year of dry storage, germination of viable achenes was more than or equal to 98%. Rudbeckia hirta achenes harvested in mid-August were nondormant and had more than or equal to 92% of viable achenes germinate. During the yearlong study, dormancy became strongest in R. hirta after six months of stratification (less than or equal to 54% germination of viable achenes) followed by a loss of dormancy one year later (more than or equal to 89% germination of viable achenes). In all three species, dormancy break was characterized by maximum germination of viable achenes, and a rapid germination (within 7 days).
Subject: Asteraceae, coreopsis, dormancy, gaillardia, germination, Rudbeckia, wildflowers
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 156 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.)--University of Florida, 2004.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0006980:00001

Permanent Link: http://ufdc.ufl.edu/UFE0006980/00001

Material Information

Title: An Ecologically based study of germination requirements and dormancies in three commercially produced Florida native wildflowers
Physical Description: Mixed Material
Language: English
Creator: Kabat, Steven Matthew ( Dissertant )
Dehgan, Bijan ( Thesis advisor )
Norcini, Jeffrey ( Reviewer )
Gordon, Doria ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2004
Copyright Date: 2004

Subjects

Subjects / Keywords: Horticultural Sciences thesis, M.S.
Dissertations, Academic -- UF -- Horticulture
Spatial Coverage: United States--Florida

Notes

Abstract: Three Florida Asteraceae species were examined for one year to determine germination parameters and presence of dormancy. Coreopsis leavenworthii, Gaillardia pulchella, and Rudbeckia hirta achenes were stratified under ambient Florida temperatures or stored dry at room temperature for one year. Achenes were germinated each month at eight different fluctuating temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/33⁰C) in the light or dark. Germination was recorded at 7, 14, and 21 days, and germinated achenes removed. Nongerminated achenes were tested for viability using 2,3,5-triphenyltetrazolium chloride. Both the June harvested C. leavenworthii and the August harvested G. pulchella achenes exhibited a physiological dormancy followed by a secondary dormancy. Late season harvested R. hirta achenes were nondormant and this was followed by a secondary physiological dormancy. Coreopsis leavenworthii's primary physiological dormancy was broken after 4 to 6 months of stratification, which resulted in more than or equal to 96% germination of viable achenes in all but one (23/33⁰C dark) treatment. For the late season harvested G. pulchella, the mild primary physiological dormancy was broken after one month of stratification, which resulted in more than or equal to 87% germination of viable achenes. After dormancy broke, most G. pulchella achenes became inviable. The achenes that remained viable entered a secondary dormancy (approximately less than or equal to 15%). After one year of dry storage, germination of viable achenes was more than or equal to 98%. Rudbeckia hirta achenes harvested in mid-August were nondormant and had more than or equal to 92% of viable achenes germinate. During the yearlong study, dormancy became strongest in R. hirta after six months of stratification (less than or equal to 54% germination of viable achenes) followed by a loss of dormancy one year later (more than or equal to 89% germination of viable achenes). In all three species, dormancy break was characterized by maximum germination of viable achenes, and a rapid germination (within 7 days).
Subject: Asteraceae, coreopsis, dormancy, gaillardia, germination, Rudbeckia, wildflowers
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 156 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.)--University of Florida, 2004.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0006980:00001


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AN ECOLOGICALLY BASED STUDY OF GERMINATION REQUIREMENTS AND
DORMANCIES INT THREE COMMERCIALLY PRODUCED FLORIDA NATIVE
WILDFL OWERS














By

STEVEN MATTHEW KABAT


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2004



























Copyright 2004

by

Steven Kabat




























This document is dedicated to my wife and our parents.
















ACKNOWLEDGMENTS

First, I would like to thank the Florida Department of Transportation (FDOT)

Environmental Management Offce for the generous funds provided for this research. I

would additionally like to thank the FDOT and the Florida Federation of Garden Clubs

for their integral role in initiating the Florida Wildflower License Plate that allowed the

citizens of Florida to voluntarily fund their roadside beautification.

I would like to thank my supervisory committee for all of their guidance. I thank

Dr. Bijan Dehgan, my supervisory committee chairman, for all his support and

encouragement. Without his help and confidence in me, I would not have been able to

complete this master' s proj ect, and for this I will always be grateful and appreciative.

Much more invaluable assistance came from my other committee members, Dr. Jeffrey

Norcini and Dr. Doria Gordon. I thank Dr. Jeffrey Norcini for his knowledge of seed

biology and his guidance during my pursuit of knowledge. I thank Dr. Doria Gordon for

always making time for me in her busy schedule and all her understanding. All my

committee members have been extremely willing to help and have enabled me to succeed

during my graduate career.

I would also like to thank the laboratory crew that made my time more productive

and efficient. I thank Fe Almira for all her help in my day-to-day scheduling of activities

and her generous, kind nature. I also thank Katherine Turner for her skillful execution of

viability testing and seed counting that was tediously performed by hand. I also

appreciate her cheerfulness and could not have asked for a better laboratory assistant.









I also thank Fred Bennett for all his help teaching me the workings of the scanning

electron microscope. I would also like to thank Marinela Capanu for her voluntary

assistance in the statistical analysis of my proj ect.

I am so grateful for the love and support of my family, especially my parents,

Richard Kabat and Joan Kabat. Both have given me the freedom to make my own

decisions and have supported me in those decisions. They have always stressed the value

of education and of hard work, and for this I am grateful. I would especially like to thank

my mother who has been a wonderful guiding force in my life. I would also like to thank

my father- and mother-in-law Terrell and Colleen Touchton. They have treated me as a

son and have supported their daughter and me in all we do. I could not have asked for a

better second family. Finally I would like to thank my wife, Cathleen Kabat, who has

played a crucial role in my education and research. She has encouraged me to work up to

and beyond what I had believed my potential to be. I also thank her for all the time she

spent working on this proj ect.





















TABLE OF CONTENTS

page


ACKNOWLEDGMENT S .............. .................... iv


LI ST OF T ABLE S ............ ......__ .............. viii..


LI ST OF FIGURE S .............. ...............x.....


AB STRAC T ................ .............. xii


CHAPTER


1 INTRODUCTION ................. ...............1.......... ......


Importance of Seed Origin............... ...............2.
Genera ................. ...............4.................
Coreopsis ................. ...............4.................
G a illllllllla rda .............. ...............6.....
Rudbeckia .............. ..... ......... ..........
Germination and Dormancy in the Asteraceae .................. .............................13
Coreopsis Germination and Dormancy Characteristics .............. ....................17
Coreopsis leavemrorthii............... .............1
Coreopsis ba~salis .............. ...............18....
Coreopsis bigelovii............... ...............1
Coreopsis lanceolata.................... .........1
Coreopsis palmata................. ...............22....... ......
Coreopsis rosea ................. ......._ ..........23... .....
Coreopsis tinctoria ................. ............ .. .. ...............23.....
Gailllllllllarda Germination and Dormancy Characteristics ................. ................ .24
Rudbeckia Germination and Dormancy Characteristics ................. ................ .25


2 MATERIALS AND METHODS .............. ...............27....


Coreopsis leavemrorthii Origin............... ...............27.
Gailllllllllarda pulchella Origin ................ ...............27........... ....
Rudbeckia hirta Origin .............. ...............28....
Species Treatment............... ...............2
Statistical Analy sis............... .. ...............3
S canning Electron Micro graph ................. ...............32........_. ....


3 RE SULT S .............. ...............34....












2001 Coreopsis leavenworthii ........._._.. ...... ...............34..
2002 Coreopsis leavenworthii ........._._.. ...... ...............36..
2002 Gaillardia pulchellla.......................3
2002 Rudbeckia hirta..........._.._. ...._... ...............46....


4 DISCUS SION AND CONCLUSIONS .............. ...............54....


Coreopsis leavenworthii ........._._ ...... .__ ...............54....
Gailllllllllarda pulchella. ................. ...............60.................
Rudbeckia hirta............... ...... .... .................6
Triphenyltetrazolium Chloride Testing Technique .............. ...............68....
Implications for Roadside Plantings ......____ .........___ ..... ............6
Coreopsis leavenworthii (mid-June Harvest) ................. ......... ................69
Gailllllllllarda pulchella (Late Season harvest) .....__.___ ..... ... ._ ........_.......70
Rudbeckia hirta (Late Season harvest) ........._.._. ......__ ......._.. ........70


APPENDIX


A Coreopsis leavenworthii FIGURES .............. ...............72....


B Gailllllllllarda pulchella FIGURES ................. ...............105...............

C Rudbeckia hirta FIGURES .........._.._.._ ...............122...._._ ....


REFERENCES .............. ...............139....


BIOGRAPHICAL SKETCH ........._..._.._ ...............143._.._._ ......

















LIST OF TABLES


Table pg

3-1 Main and interactive effects of temperature, light, and months of stratification on
germination of viable achenes of C. leavenworthii harvested June 19, 2002. .........34

3-2 Main and interactive effects of temperature, light, and months of stratification on
germination of viable achenes of G. pulchella harvested August 12, 2002.............3 5

3-3 Main and interactive effects of temperature, light, and months of stratification on
germination of viable achenes of R. hirta harvested August 9, 2002. .....................35

3-4 Maximum percent germination (n = 4 + SD) of total 2001-harvested C.
leavenworthii achenes with statistically insignificant months for each treatment...37

3-5 Maximum percent germination (n = 4 + SD) of total 2002-harvested C.
leavenworthii achenes with statistically insignificant months
for each treatment. .............. ...............40....

3-6 Percent germination (n = 4 + SD) of freshly harvested and 14-month dry stored
(DS) viable C. leavenworthii achenes harvested in 2002............... ..................4

3-7 Percent viability (n = 4 + SD) of freshly harvested and 14-month dry
stored (DS) C. leavenworthii achenes harvested in 2002............... ..................4

3-8 Maximum percent germination (n = 4 + SD) of total 2002-harvested G. pulchella
achenes with statistically insignificant months for each treatment. .........................44

3-9 Percent germination of viable (n = 4 + SD) freshly harvested and 12-month dry
stored (DS) G. pulchella achenes harvested in 2002. ............. .....................4

3-10 Percent viability (n = 4 + SD) of freshly harvested and 12-month dry stored (DS)
G. pulchella achenes harvested in 2002. ............. ...............47.....

3-11 Maximum percent germination (n = 4 + SD) of stratified and 12-month dry
stored (DS) G. pulchella achenes harvested in 2002. ............. .....................4

3-12 Maximum percent germination (n = 4 + SD) of total 2002-harvested R. hirta
achenes with statistically insignificant months for each treatment. .........................51










3-13 Percent germination of viable (n = 4 + SD) freshly harvested and 12-month dry
stored (DS) R. hirta achenes harvested in 2002 for each treatment. ........................52

3-14 Percent viable (n = 4 + SD) of freshly harvested and 12-month dry stored
(DS) viable R. hirta achenes harvested in 2002 for each treatment. ........................53

















LIST OF FIGURES


Figure pg

1-1 Scanning electron micrograph of a cross-section of a C. leavenworthii achene at
250X magnification ................. ...............7.................

1-2 Scanning electron micrograph of an intact C. leavenworthii achene at 35X
magnification ................. ...............8.................

1-3 Scanning electron micrograph of the pappus of an intact C. leavenworthii achene at
150X magnification ................. ...............9............ ....

1-4 Scanning electron micrograph of a G. pulchella achene at 30X magnification of an
achene longitudinal-section with the aristate pappus scales present. ....................10

1-5 Scanning electron micrograph of G. pulchella achene at 40.6X magnification of the
exterior of an intact achene with the basal tuft of trichomes evident. ................... ...1 1

1-6 Scanning electron micrograph of R. hirta achene at 45X magnification of the
exterior of an intact achene with evident longitudinal ribbing along the length
of the linear cylindrical body. ............. ...............12.....

1-7 Scanning electron micrograph ofR. hirta achene at 50X magnification of an
achene longitudinal-section with conspicuous embryo.............__ ..........__ .....13

2-1 Mean minimum, maximum, and monthly temperatures (oC) for Gainesville, FL,
recorded from 1951 through 1980 (SCS 1985). ................... ............... 3

2-2 Extreme high, extreme low, and monthly mean precipitation (cm) for Gainesville,
FL, recorded from 1951 through 1980 (SCS 1985). ............. ......................1

3-1 Germinated R. hirta achenes harvested after 4 months of stratification in their cloth
stratification envelope. ............. ...............50.....

A-1 Mean (n = 4) percent germination (+ SD) of total 2001-harvested C.
leavenworthii achenes. ............. ...............72.....

A-2 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2001-
harvested C. leavenworthii achenes at a 12 hr photoperiod .................. ...............76










A-3 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2001-
harvested C. leavenworthii achenes in complete darkness. ............. ...................80

A-4 Mean (n = 4) percent germination (+ SD) of total 2002-harvested C.
leavenworthii achenes. ............. ...............84.....

A-5 Mean (n = 4) percent germination (+ SD) of viable 2002-harvested C.
leavenworthii achenes. ............. ...............88.....

A-6 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-
harvested C. leavenworthii achenes at a 12 hr photoperiod .................. ...............92

A-7 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-
harvested C. leavenworthii achenes in complete darkness. ............. ...................96

A-8 Mean (n = 4) percent viability (+ SD) of 2002-harvested C.
leavenworthii achenes. ............. ...............100....

B-1 Mean (n = 4) percent germination (+ SD) of viable 2002-harvested G. pulchella
achenes. ............. ...............105....

B-2 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-
harvested G. pulchella achenes at a 12 hr photoperiod. ............_.._ .........._.... ...109

B-3 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-
harvested G. pulchella achenes in complete darkness. ................ ................ ...113

B-4 Mean (n = 4) percent viability (+ SD) of 2002-harvested G. pulchella achenes. ..1 17

C-1 Mean (n = 4) percent germination (+ SD) of viable 2002-harvested R.
hirta achenes. ............. ...............122....

C-2 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-
harvested R. hirta achenes at a 12 hr photoperiod. ............. ......................2

C-3 Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2002-
harvested R. hirta achenes in complete darkness. ................. ..................3

C-4 Mean (n = 4) percent viability (+ SD) of total 2002-harvested
R. hirta achenes. ............. ...............134....
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

AN ECOLOGICALLY BASED STUDY OF GERMINATION REQUIREMENTS AND
DORMANCIES INT THREE COMMERCIALLY PRODUCED FLORIDA NATIVE
WILDFLOWERS

By

Steven Matthew Kabat

August 2004

Chair: Bijan Dehgan
Major Department: Environmental Horticulture

Three Florida Asteraceae species were examined for one year to determine

germination parameters and presence of dormancy. Coreopsis leavenworthii, Gailllllllllllllllllari

pulchella, and Rudbeckia hirta achenes were stratified under ambient Florida

temperatures or stored dry at room temperature for one year. Achenes were germinated

each month at eight different fluctuating temperature regimes (5/20, 10/20, 10/25, 15/25,

15/30, 20/25, 20/30, and 23/330C) in the light or dark. Germination was recorded at 7,

14, and 21 days, and germinated achenes removed. Nongerminated achenes were tested

for viability using 2,3,5 -triphenyltetrazolium chloride. Both the June harvested C.

leavenworthii and the August harvested G. pulchella achenes exhibited a physiological

dormancy followed by a secondary dormancy. Late season harvested R. hirta achenes

were nondormant and this was followed by a secondary physiological dormancy.









Coreopsis leavemrorthii's primary physiological dormancy was broken after 4 to 6

months of stratifieation, which resulted in >96% germination of viable achenes in all but

one (23/330C dark) treatment. For the late season harvested G. pulchella, the mild

primary physiological dormancy was broken after one month of stratifieation, which

resulted in >87% germination of viable achenes. After dormancy broke, most G.

pulchella achenes became inviable. The achenes that remained viable entered a

secondary dormancy (approximately <15%). After one year of dry storage, germination

of viable achenes was >98%. Rudbeckia hirta achenes harvested in mid-August were

nondormant and had 192% of viable achenes germinate. During the yearlong study,

dormancy became strongest in R. hirta after six months of stratifieation (554%

germination of viable achenes) followed by a loss of dormancy one year later (189%

germination of viable achenes).

In all three species, dormancy break was characterized by maximum germination of

viable achenes, and a rapid germination (within 7 days).















CHAPTER 1
INTRODUCTION

President Clinton issued an Executive Memorandum on Environmentally and

Economically Beneficial Landscape Practices on Federal Landscaped Grounds on April

26, 1994 to encourage states to work toward the following goals: use regionally native

plants in public landscapes, minimize detrimental effects on natural habitats through

environmentally benign construction practices, prevent pollution and misuse of water and

energy, and create outdoor demonstration proj ects to inform the public (Harper-Lore and

Wilson 2000).

The Florida Wildflower License Plate initiative was implemented to fulfill the

directives of the Executive Memorandum. The Florida Department of Transportation

(FDOT), in conjunction with the Florida Federation of Garden Clubs, sponsored the

Florida Wildflower License Plate, which were to be sold in order to raise the funds

necessary for native wildflower research, educational programs, and community-based

grant programs. The research component was necessary due to several of the FDOT

districts' past experiences with planting failures and the lack of technical information

about Florida native wildflower seed production. The FDOT realized the need for

research focusing on seed germination characteristics, which would provide insight on

important aspects of successful stand establishment, including seeding rate, depth of

planting, date of seeding, and in what habitats/climates the seeds needed to be planted

(Florida Department of Transportation 2003).









Importance of Seed Origin

When trying to implement native wildflowers into roadside restoration, reclamation

and even in commercial and residential landscaping, the utilization of plant material

derived from local origins is important. Some sources suggest that plant material should

originate from a distance no greater than 160 to 320 km (100 to 200 miles) from the

planting location because locally derived plant material is often better adapted to local

conditions (greater drought tolerance and increased disease resistance); however, soil,

climate, and hydrology may be more important to plant establishment than physical

distance (Harper-Lore and Wilson 2000, Pfaff et al. 2002). Non-local plant material may

appear more attractive, but some non-local plants may perform poorly over time. Norcini

et al. (1998) compared the field performance of Florida native wildflowers Coreopsis

lan2ceolata L., Gaillardia pulchella~11~11~11~ Foug., and Rudbeckia hirta L. under low input north

Florida conditions with seed obtained from commercial producers outside Florida. They

found that the north Florida ecotype of C. lan2ceolata differed morphologically and

responded differently than plants from the seed source outside of Florida. The plants

grown from seed derived from outside of Florida did not flower at three of the five test

sites and also had an increased incidence of disease and insect damage. The G. pulchella

plants obtained from different seed sources also exhibited morphological differences.

The non-Florida plants' flowering duration and overall survival rate was noticeably

shorter when compared to the north Florida ecotype. Rudbeckia hirta also exhibited

distinct morphological and flowering differences between seed sources (Norcini et al.

1998).

It has also been suggested that species inhabiting large geographic ranges and thus

exposed to varied climatic conditions may have germination requirements adapted to the









selection pressures of the region of origin (Harper-Lore and Wilson 2000). Another

reason to use local ecotypes is to prevent the spread of genetic material (also referred to

as "genetic pollution") from crops originating from outside the local area or from

horticulturally manipulated crops (Pfaff et al. 2002). Wild-crop Fl hybrids of the

common sunflower, Helianthus annuus L. (Asteraceae), have changed many population

characteristics of the native species (Snow et al. 1998). The cultivated varieties were

bred to have strong apical dominance and to be devoid of dormancy. Fl wild-crop

hybrids have exhibited decreased flower-head production and branching. These hybrids

also have decreased achene production and reduced achene dormancy.

Two of the study species, G. pulchella and R. hirta, that are commercially available

in Florida are produced in other areas of the country. Both are commonly available in

seed catalogs and are used in gardens across the country. Coreopsis leavenworthii Torr.

& A. Gray has been available to the public in the large chain stores in Florida since 1999

as part of Riverview Flower Farm' s Florida Friendly PlantsTM line.

In this study, Florida ecotypes of C. leavenworthii, G. pulchella, and R. hirta, were

studied in order to determine natural germination parameters and to examine the effect

dormancy, if present, had on those germination parameters. This was done by artificial

simulation of natural and commercial storage conditions followed by laboratory

germination testing at typical Florida seasonal temperatures. Moisture-controlled

stratification of achenes was conducted under ambient Florida temperatures in order to

simulate the natural environment. The commercial storage technique of a climate-

controlled dry storage was also employed. Germination testing followed storage.

Dormancy was determined from the characteristics in which germination occurred. The









information obtained was ultimately intended to aid the FDOT in their attempts to

employ successful roadside wildflower vegetation management practices.

Genera

The three genera under study (Coreopsis, Gaillardia,~ll11~~~111~~~ and Rudbeckia) are species-

rich. Because of this diversity, historical examination will be limited to germination

characteristics within the prescribed genera and to the individual species.

Coreopsis

Much work has been accomplished with the goal of determining the systematic

relationships within the genus Coreopsis (Asteraceae: Tribe Heliantheae: Subtribe

Coreopsidinae). There are 75 to 80 recognized species of Coreopsis tickseedd) native to

the Americas. The genus Coreopsis is believed to be a paraphyletic assemblage with taxa

traditionally prescribed to the genus Bidens arising at two different occasions within the

genus Coreopsis. The eastern North American species appear to form a clade that

includes, but is not limited to, all the species described below, with the exception of C.

bigelovii (A. Gray) H.M. Hall (Kim et al. 1999).

The section Calliopsis is composed of three members; C. leavenworthii, C.

tinctoria Nutt., and C. paludosa Nutt. Coreopsis leavenu11~ 1,i thii appears to be more

closely related phylogenetically to C. tinctoria than either one is to C. paludosa (Jansen

et al. 1987). Morphological evidence and occupation of the same low-elevation, moist,

ruderal habitats suggest the possibility of hybridization between C. leavenworthii and C.

tinctoria in northwestern peninsular Florida (Crawford et al. 1984; Smith 1978). These

species, however, are believed to retain distinct morphological differences through the

maj ority of their ranges and have not been suggested for species combination (Crawford

et al. 1984).









Kim et al. (1999) have proposed that section Eublepharis (C. rosea and C. gladiata

Walter) is nested within section Calliopsis (C. leavenworthii, C. tinctoria, and C.

paludosa M.E. Jones). The Eublepharis + Calliopsis clade is sister to section Coreopsis

(C. ba~salis [Dietr.] Blake, C. grandiflora Hogg ex Sweet, C. lan2ceolata, and relatives).

Section Palmatae (C. palmate Nutt. and relatives) is a member of a clade that also

contains the above listed groups along with section Silphidium and several species of

Bidens. Coreopsis bigelovii (Section Pugiopappus) is the most distantly related to C.

leavenworthii and is not even included in the eastern North American clade (Kim et al.

1999).

In Florida, there are 13 species of Coreopsis found in the wild. Of these, C.

floridanadd~~~~~ddddd~~~~ E.B. Sm. and C. leavenworthii are endemic, and some scientists believe C.

ba~salis and C. tinctoria have naturalized in Florida from other areas of the United States

(Wunderlin and Hansen 2003).

Coreopsis leavenworthii (Leavenworth's tickseed) is an herbaceous annual to short-

lived perennial. Its flowers are aggregated into capitate heads that are less than 5 cm in

diameter on long peduncles in open corymbs (Ledin 1951). The heads are composed of

sterile yellow ray florets and fertile dark brown disk florets. The floral heads are

observable from mid-spring to fall in northern Florida to all year in southern Florida, but

are particularly abundant in spring (Taylor 1998). The fruit is a thin oval achene (Fig. 1-

1) with two tan lateral wings. Each wing is as broad as the achene's dark brown body,

which contains the embryo (Fig. 1-2). The achene also has a pappus of two short awns

(Fig. 1-3) (Ledin 1951).









Coreopsis leavemrorthii' s range covers most of Florida, but has not been

vouchered in Calhoun, Clay, Escambia, Gulf, Hamilton, Holmes, Leon, Liberty,

Madison, Nassau, or Okaloosa Counties, probably due to under-collection. It has been

documented in floras as far back as 1887 (Chapman 1887). The oldest herbarium

specimen at the University of Florida is from Tampa, Hillsborough County in 1876.

Coreopsis leavemrorthii grows in wet pine flatwoods, glades, prairies, and various

ruderal sites, including roadside ditches and, rarely, in dry pinelands (Ledin 1951; Taylor

1998). Pine flatwoods, which comprise 50 percent of the land area in Florida, are

characterized by low, flat topography and sandy, acidic soil underlain by a clay hardpan

(which restricts drainage and often creates a seasonally high water table). Prior to

European settlement, frequent fires naturally maintained pine flatwoods. The largest

diversity of Florida wildflowers occurs in open-canopy flatwoods where fire is frequent

(Myers and Ewel 1990).

Gaillardia

The genus Gailllllllllllllllllarda (Asteraceae: Tribe Heliantheae) is represented in Florida by

two species: G. aestivalis (Walter) H. Rock and G. pulchella. Gailllllllllllllllllardapulcel is

commonly known as Blanket Flower, Indian Blanket, or Firewheel (Turner and Whalen

1975; Wunderlin and Hansen 2003). It has been documented nearly throughout Florida

and is often used as an ornamental that tends to freely reseed outside of its original

planting site (Bell and Taylor 1982; Turner and Whalen 1975; Wunderlin and Hansen

2003). The species range covers most of the United States, excluding the northwestern

states, west of South Dakota and Minnesota (Niering and Olmstead 2001). Gailllllllllllllllllarda

pulchella is a halophyte (salt-loving plant) but is not limited to coastal sites, also





































Figure 1-1. Scanning electron micrograph of a cross-section of a C. leavemrorthii achene
at 250X magnification. The cross-section was made perpendicular to the
achene's wings. The bar in the figure is equivalent to 0.12 mm. The image
was recorded on a Hitachi 4000 FESEM at the Interdisciplinary Center for
Biotechnology Research in the Electron Microscopy Core Laboratory,
University of Florida.

inhabiting prairies, sandy open sites, and roadsides (Niering and Olmstead 2001). In

coastal ecosystems, G. pulchella contends with shifting sand, sand abrasion, intense

sunlight, nutrient-poor soils, salt spray, and desiccation (Taylor 1998). Turner and

Whalen (1975) recognize three varieties (picta, australis, and pulchella), but I will refer

to the species as a whole, as per Wunderlin and Hansen (2003).

Gaillardia pulchella~11~11~11~ is an annual to short-lived perennial. Its peak bloom time in Florida

occurs from May through August, but bloom may occur any time of year (Wunderlin and

Hansen 2003). Flowerheads are born terminally, with 6 to 15 ray florets. The ray florets




































Figure 1-2. Scanning electron micrograph of an intact C. leavenworthii achene at 35X
magnification. The achene's two prominent wings are evident. The bar in the
figure is equivalent to 1 mm. The image was recorded on a Hitachi 4000
FESEM at the Interdisciplinary Center for Biotechnology Research in the
Electron Microscopy Core Laboratory, University of Florida.

may be entirely red, entirely yellow, or reddish purple with yellow notched apices (Bell

and Taylor 1982). The center of the head is composed of fertile reddish purple disk

florets. Floral heads range from 2.5 to 7.5 cm in diameter (Taylorl998). Achenes are

born on a receptacle containing firm, subulate (awl-shaped) setae. Achenes have 6 to 10

aristate (stiff apical bristle or awn) pappus scales (Fig. 1-4) and a conspicuous basal tuft

of trichomes (Fig. 1-5) (Turner and Whalen 1975; Wunderlin and Hansen 2003).

Gaillardia pulchella~11~11~11~ is a diploid, obligate outcrosser (self-incompatible) (Heywood

1993).





































Figure 1-3. Scanning electron micrograph of the pappus of an intact C. leavemrorthii
achene at 150X magnification. The achene's pappus is composed of two short
awns. The bar in the figure is equivalent to 0.2 mm. The image was recorded
on a Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology
Research in the Electron Microscopy Core Laboratory, University of Florida.

Rudbeckia

The genus Rudbeckia is in the Helianthus tribe in the Asteraceae. There are about

25 species native to North America. There are nine Rudbeckia taxa in Florida, including

R. hirta, commonly known as Black-eyed Susan. Rudbeckia hirta occurs throughout

North America, except for Arizona, Nevada, and the far north (Harkess and Lyons 1994).

In Florida, R. hirta commonly grows in sandhills, pine flatwoods, and open disturbed

sites and is found nearly throughout the state (Wunderlin and Hansen 2003).

Rudbeckia hirta is an annual to short-lived perennial. In Florida, flowering

primarily occurs from April through October. The flowerheads contain yellow ray florets











Y:
I
I r'r$1!
:' ?~~*~g


Figure 1-4. Scanning electron micrograph of a longitudinally sectioned G. pulchella
achene at 30X magnification with the aristate pappus scales present. The bar
in the figure is 1 mm. The image was recorded on a Hitachi 4000 FESEM at
the Interdisciplinary Center for Biotechnology Research in the Electron
Microscopy Core Laboratory, University of Florida.

and purple brown disk florets that are attached to a chaffy conical receptacle. The disk

florets are fertile and have divided styles (Harkess and Lyons 1994; Niering and

Olmstead 2001; Norcini personal communication). The actinomorphic heads measure

3.8 to 7.5 cm across (Niering and Olmstead 2001; Norcini et al. 2001). Achenes are dark

brown and linear with longitudinal ribbing along the length of the cylindrical body (Fig.


1-6) that contains the embryo (Fig. 1-7).

As was mentioned in the species description, R. hirta covers much of the eastern

United States. Marois and Norcini (2003) investigated regional differences in the species

in a field trial conducted in Quincy, Florida. They found significantly lower season-long





































Figure 1-5. Scanning electron micrograph of the exterior of a G. pulchella achene at
40.6X magnification with the basal tuft of trichomes evident. The bar in the
figure is 1 mm. The image was recorded on a Hitachi 4000 FESEM at the
Interdisciplinary Center for Biotechnology Research in the Electron
Microscopy Core Laboratory, University of Florida.

survival from a Texas source as compared to those attained from northern Florida and

central Florida seed sources, although they could not attribute the difference in plant

survival to fungal attack, irrigation, or climate (precipitation or temperature).

Consequently, seed source was the only attributable difference, but they recognized the

need for further study into the differences in survival. It was also hypothesized that the

cause could have been due to the Texas material coming from a shorter-season

population as compared to Florida (Marois and Norcini 2003). Another study by Norcini

et al. (2001) examined latitudinal site variability (four sites within Florida) on plant

growth, flowering, and survival among two Florida ecotypes and one from Texas.




































Figure 1-6. Scanning electron micrograph of an intact R. hirta achene at 45X
magnification with evident longitudinal ribbing along the length of the linear
cylindrical body. The bar in the figure is 1 mm. The image was recorded on a
Hitachi 4000 FESEM at the Interdisciplinary Center for Biotechnology
Research in the Electron Microscopy Core Laboratory, University of Florida.

Despite similarities in the soil texture, the sites had different rainfall amounts, nutrient

levels, and degrees of nematode infestations. Collectively, the environmental differences

somewhat obscured the results. Survival was significantly higher for the Florida

ecotypes in three of the four sites compared to the Texas ecotype, despite site variability.

The fourth site showed no variability among ecotypes, but the plants had an unusually

short lifespan compared to the other sites (Norcini et al. 2001).

Rudbeckia hirta has been used as a model species in the study of wildflower sod

production. Neigebauer et al. (2000) examined three mowing heights, 5.1 cm, 7.6 cm



































Figure 1-7. Scanning electron micrograph of a longitudinally sectioned R. hirta achene at
50X magnification with the conspicuous embryo present. The bar in the
figure is 1 mm. The image was recorded on a Hitachi 4000 FESEM at the
Interdisciplinary Center for Biotechnology Research in the Electron
Microscopy Core Laboratory, University of Florida.

(the height typically used in wildflower sod production), and 10.2 cm, and compared

them to plant material that was not mowed. The study concluded that, as mowing height

was increased from 5.1 cm to a non-mowed condition, there was a corresponding

increase in root dry-weight, rooting depth, and the amount of root axes in the top 2.5 cm

of soil, the layer typically harvested in production.

Germination and Dormancy in the Asteraceae

Nondormant seeds that do not germinate for reasons other than a lack of sufficient

external moisture or unfavorable environmental conditions are not necessarily dormant,

they may just be quiescent. Quiescent seeds sometimes have been referred to as having

an enforced dormancy or said to have an environmental inhibition to germination (Baskin









and Baskin 2001). An environmental factor such as available moisture or oxygen

(excluding seed's physical structure) able to be changed may enhance germination by

ending quiescence. Quiescent seeds are non-dormant seeds in which metabolism is

reduced or halted due to an environment that is below or above that required for optimal

germination. Dormant seeds are unable to germinate even after exposure to optimal

germinating conditions, while quiescent seeds will germinate upon exposure to optimal

conditions (Baskin and Baskin 2001). In addition, dormant seeds have one or more

barriers to overcome before germination can occur and these barriers collectively make

up a seed' s dormancy (Baskin and Baskin 2001).

Primary (innate or conditional) dormancy occurs when seeds are dormant directly

after they are released from the mother plant. Physiological dormancy (PD) is a type of

primary dormancy and is endogenous or embryo-based with germination inhibited by a

physiological mechanism. There are three types of PD: nondeep, intermediate, and deep.

Nondeep PD is characterized by recently matured seeds that only germinate over a

narrow temperature range (referred to as conditional dormancy or relative dormancy) or

by a lack of germination at any temperature (primary innate dormancy). Seeds with

nondeep PD are the only seeds that have the ability to annually cycle between dormancy

and nondormancy. Such seeds go through a state of conditional dormancy during the

transitional period. This cycling is gradual and occurs in response to environmental cues.

Some seeds only cycle between conditional dormancy and nondormancy (Baskin and

Baskin 2001; Bewley and Black 1994).

The literature refers to afterripening in two different ways. It is refers to as the

process dry stored seeds undergo as they become nondormant and it is also referred to as









the duration storage (typically dry) as a dormancy breaking technique (Baskin, Baskin,

and Van Auken 1992; Bewley and Black 1994). The latter definition will be used in this

document. Afterripening occurs in dry stored seeds, but the seeds themselves may have

up to 20% water content. There are three different response patterns associated with

dormancy break. During the type-1 response, the maximum temperature limit at which

germination may occur increases as conditional dormancy is broken. In a type-2

response, a seed's minimum temperature at which germination may occur decreases. The

type-3 response is characterized by both the upper and lower germination temperature

limits increasing and decreasing, respectively. Of the 32 Asteraceae species whose

response process had been studied by Baskin, Baskin, and Van Auken (1992), three were

type-1, 22 were type-2, and seven were type-3. The type-1 response pattern is usually

found in species with a winter life cycle. Both the limited number of species studied and,

possibly, the small number of species that experience a winter life cycle may be the

hypothesized explanation for the limited number of species encountered with the type-1

response pattern (Baskin, Baskin, and Van Auken 1992).

Secondary (induced) dormancy occurs when mature imbibed seeds experience sub-

optimal or super-optimal environmental conditions (anaerobic environment, too much

darkness or light, temperatures excessively high or low, or water stress) that trigger

dormancy. Similar to primary dormancy, secondary dormancy may be innate or

conditional. Secondary dormancy often begins as a seed coat imposed dormancy and

then develops into a condition of embryo dormancy (Baskin and Baskin 2001; Bewley

and Black 1994).









Another sign that seeds may have a nondeep PD is the presence of a light

requirement that may or may not be lost as dormancy is broken (Baskin and Baskin

2001). Also, seeds that have a nondeep PD can achieve normal embryo growth when the

embryo is isolated from the seed. Barriers associated with nondeep PD are usually

attributed to the interaction between the embryo and its covering structures. The

covering structures may influence dormancy by: controlling oxygen passage to the

embryo, controlling the movement of growth inhibitors away from the embryo, or

physically restricting the embryo's growth potential by controlling enzymatic breakdown

of embryo covering structures. Nondeep PD may be broken through the application of

exogenous chemicals, including kinetin, gibberellins, ethylene, and potassium nitrate

(KNO3), that effectively substitute for specific environmental dormancy breaking

conditions (Baskin and Baskin 2001).

An intermediate PD is characterized by the need for an increased duration (< 6

months) of dormancy-breaking treatment (mainly stratification) prior to germination.

The intermediate PD's dormancy-breaking treatment may be shortened by room-

temperature dry storage or by the application of exogenous chemicals. Isolated embryos

with an intermediate PD can also achieve normal embryo growth upon removal from the

seed like that of a nondeep PD.

Deep PD is differentiated by the requirement of a long period of dormancy-

breaking treatment for intact dispersal units; it cannot be shortened by implementing any

of the aforementioned techniques and isolated embryos do not develop normally (Baskin

and Baskin 2001).









There are various other types of dormancies that may occur in combination with

physiological dormancy. When time is required for growth of an underdeveloped or

undifferentiated embryo prior to germination, it is a type of endogenous dormancy

termed morphological dormancy (Baskin and Baskin 2001). Physical dormancy is the

result of a water impermeable fruit or seed coat. Chemical dormancy prevents or inhibits

germination by chemical inhibitors usually located in the pericarp. The inhibitors need to

be leached or physically removed before chemical dormancy is broken and germination

can occur. Abscisic acid is commonly associated with chemical dormancies. However, it

is not known to what degree it is responsible for the cause of a chemical dormancy.

Mechanical dormancy (coat-imposed dormancy) is a restriction of growth due to a woody

fruit wall usually formed from the endocarp and/or mesocarp or the endosperm. In a

mechanical dormancy the surrounding structures restrict growth of the embryo until

dormancy is broken. All of the above types of dormancies may occur separately or in

combination with one another.

Many members of the Asteraceae experience physical dormancy due to a semi-

permeable inner membranous seed coat that has the ability to retard water and oxygen

exchange with the embryo. The semi-permeable membrane may also prevent leaching of

germination inhibitors located within the cotyledons (Atwater 1980). Some species of

Asteraceae, including tickseeds (Coreopsis), also may have a waxy covering on the seed

coat that may prevent water and/or gas exchange (Voigt 1977).

Coreopsis Germination and Dormancy Characteristics

Coreopsis leavenworthii

Beyond the morphological species description, distribution range in Florida, and

growth habitats, little information is available about C. leavemrorthii, especially









regarding its biology or ecology. Norcini and Aldrich (2003) examined sowing date on

six Florida wildflowers. Plots (15 ft2) were sown with 2001 and 2002 harvested C.

leavemrorthii achenes during the first week of each month from July through December

2002 and evaluated on March 24, 2003. The largest number of plants was achieved from

the September sowing during both harvest years. The September sowing also visually

rated well when judged on flowering, wildflower density, and weediness. The more

recently harvested 2002 achenes had equivalent or higher number of established plants

throughout testing. The 2002 harvest also had good plant establishment between August

and November.

Coreopsis basalis

Coreopsis basa~lis is found from Florida' s central peninsula north into the

panhandle in scattered areas (Wunderlin and Hansen 2003). The Association of Official

Seed Analysts (AOSA) standardized germination test for C. basa~lis is performed by

placing the achenes on top of a blotter at a constant 200C and checking at 8 days (AOSA

1994).

Coreopsis bigelovii

Coreopsis bigelovii is a Moj ave Desert annual that, in its native environment,

exhibited dormancy after dispersal from the mother plant. Capon and Van Asdall (1967)

used pre-germination heat treatment to break dormancy; greatest germination (~30%)

was attained at 200C after 8 weeks or at 500C after 5 weeks, although a storage time of 1

week at 500C yielded 26% germination. Germination of freshly harvested seed was only

9% (Capon and Van Asdall 1967).









Coreopsis lanceolata

In Florida, C. lan2ceolata can be found growing in sandhills and disturbed sites

from northern Florida southward to Lake County (Wunderlin and Hansen 2003). Its

broad geographic range extends west to Texas and New Mexico, and north to Lake

Superior (Banovetz and Scheiner 1994a; Banovetz and Scheiner 1994b).

Germination tests for C. lan2ceolata have been standardized, but recommended

guidelines for optimum germination practices vary throughout the literature. For the

AOSA protocol (1994), achenes are germinated on top of a blotter at an alternating

20/300C (or at a constant 150C) in light and germination is checked at 7 and 21 days.

The blotters should be moistened with a 0.2% solution of KNO3. Light should be

provided during the high temperature and for a minimum of 8 hr. The lower temperature

should be held constant for 16 hr followed by 8 hr at the higher temperature (AOSA

1994). Atwater (1980) found that immersing achenes in water followed by immersion in

0.2% KNO3 improved germination at 150C for 40 days. Carpenter and Ostmark (1992)

tested the germination characteristics of achenes collected from north-central Florida in

May when the achenes were naturally dispersing. Germination tests were conducted at a

constant 150C on a double layer of filter paper substrate in Petri dishes wetted with

distilled water. The 150C optimal temperature for germination tests was determined after

comparison between different constant and alternating temperatures. The effect of

growth regulators was also studied. A 6 hr treatment of 1,000 ppm ethephon plus 1,000

ppm gibberellic acid (GA3) significantly improved germination of freshly harvested

achenes. After 21 days germination test, 79% of the seeds germinated, with half of them

germinating within 6. 1 days of the start of the test and the maj ority of them over a 5.5-

day period. Single ethephon and GA3 growth regulator treatments also significantly










improved germination over the control but not to the same degree as the combination

treatment. The duration of fresh seed storage was also examined for different relative

humidities. Optimal germination (80%) was attained at 150C after 7 months of storage at

20 to 35% relative humidity (Carpenter and Ostmark 1992). Banovetz and Scheiner

(1994a) determined that optimal germination could be attained at 15 or 250C under a 12

hr photoperiod.

Banovetz and Scheiner (1994a) also reported that germination percentages of C.

lan2ceolata increased as the dry achenes aged from 2 to 20 months; the percentages

declined thereafter. This increase in germination percentage was probably a result of

afterripening. This primary (innate) dormancy is usually attributed to achene structural

or physiological barriers. Coreopsis lan2ceolata was induced into a secondary dormancy

when previously dry-stored achenes were imbibed and placed at SoC for increasing

lengths of time and then moved to 24 hr of light. As exposure time at SoC increased,

subsequent germination percentage decreased, indicating the existence of a secondary

dormancy. The dry-stored achenes that entered into the secondary dormancy could not

be brought out of it even with subsequent light/dark cycles, freezing temperatures, or

warmer temperatures (Banovetz and Scheiner 1994a).

The effect of vegetation cover has been studied with respect to the percentage

germination of C. lan2ceolata in the field. It was concluded that seedlings emerged in

significantly higher numbers in non-vegetated as compared to vegetated plots (Banovetz

and Scheiner 1994a).

The effect of seed mass on the ability to germinate and subsequent seedling

viability of C. lan2ceolata has also been investigated. Achenes of C. lan2ceolata that had a










greater mass had higher viability after burial and emerged from greater depths as

compared to achenes with lower masses. When predispersed achenes of a Michigan

ecotype were collected and analyzed, they displayed a traditional bell-shaped curve for

seed mass with a mean around 0.8 mg. However, when collected from a native soil seed

bank (the maj ority from 0 to 2 cm in depth), the mean of the maj ority of achenes was

below 0.4 mg (Banovetz and Scheiner 1994b). The authors suggested that this low

weight could be attributed to a combination of predation of larger achenes, germination

out of the seed bank, and the lower probability that larger-sized achenes can work their

way into the seed bank. When the natural seed bank was tested for viability, only those

achenes greater than 0.4 mg were viable. While those achenes in the seed bank less than

0.4 mg were almost 30 times more abundant, they were not viable (Banovetz and

Scheiner 1994b). As part of the same study, C. lan2ceolata achenes with a larger mass

that were manually buried for two years in the field (i.e., forced into the seed bank)

retained their viability longer than achenes with a lower mass. Field-buried achenes with

a larger mass, however, displayed lower germination rates, indicating that they had

developed dormancy. The dormancy was most likely a secondary dormancy caused by

the cold winters in Michigan (Banovetz and Scheiner 1994a).

The effect of seed priming on C. lanceolata achenes has also been studied.

Osmotic priming (osmoconditioning) is a process that controls or limits imbibition

through the use of inorganic salts (e.g., potassium phosphate) or polyethylene glycol

(PEG). This controlled imbibition enhances seed viability, improves germination speed

and uniformity, and expands the range of temperatures suitable for germination of aged

seed by slowing germination processes. The success of osmoconditioning is likely









accomplished by allowing repair of age-induced damage (Bewley and Black 1994; Fay

1994; Samfield et al. 1991). Primed achenes of C. lanceolata exhibited increased

germination percentages, rapidity, uniformity, and, ultimately, a faster growing, more

uniform crop after priming in a 50 mM potassium phosphate buffer solution and to a

slightly lesser extent after priming in distilled water. A 3- and 6-day priming treatment at

approximately 160C in both osmotica resulted in the greatest priming benefit (Samfield et

al. 1991).

Achenes of C. lan2ceolata also responded favorably to 2 months of vacuum storage

at 120C. The rate of germination and the total germination percentage was improved

over the control that was not vacuum stored. The vacuum probably reduced respiratory

activity in storage, resulting in prolonged viability (Samfield et al. 1990).

Coreopsis palmata

Coreopsis palmata was collected for a study from a northern Illinois prairie

remnant (Voigt 1977). The original seed had only 50% embryo development, but of

those that developed, 96% tested viable when a 2,3,5-triphenyltetrazolium chloride (TZ)

test was administered. The achenes in the experiment were treated with a fungicide

(Arasan@) and then germinated on moist filter paper in Petri dishes in darkness at room

temperature (~240C), although the darkness was interrupted daily for germination counts.

Achenes were probably dormant since germination was only 40% after 30 days.

Germination increased to 98% after cold stratification at 40C for 60 days. Stratification

also narrowed the timing of germination. Germination occurred between 4 and 30 days

without stratification and between 6 and 12 days with stratification.









Coreopsis rosea

Coreopsis rosea is an obligate perennial (requires two growing seasons to

reproduce) found in southeastern Canada south to Georgia. The achenes collected from

this region were cold stratified at 40C for 270 days and then germinated in Petri dishes at

an alternating temperature of 20/300C and a 15 hr photoperiod. The achenes began to

germinate after 6 days and achieved a maximum of 50% germination after 30 days

(Shipley and Parent 1991).

Coreopsis tinctoria

Coreopsis tinctoria is native to the southeastern United States, but is considered a

non-native cultivated introduction to Florida by many botanists. It may be found growing

in wet and open disturbed areas in the state (Wunderlin and Hansen 2003). The species

was not recorded as inhabiting Florida in Chapman's flora (1887) and University of

Florida herbarium specimens only date back to a 1932 Alachua County specimen and a

1948 Dade County specimen. Some roadsides in northern Florida have been

intentionally seeded with this species by the FDOT and possibly other agencies

responsible for roadside vegetation management. The species is regarded as a winter

annual weed in temperate grasslands (Baskin and Baskin 2001).

The fruit and seed structures have been investigated in great detail (Pandey and

Singh 1982). The AOSA protocol (1994) for C. tinctoria requires achenes to be placed

on top of a blotter at a constant 200C and germination to be recorded at 8 days. However,

Kaspar and McWilliams (1982) reported that optimal germination of C. tinctoria was at

constant 300C under continuous illumination. They did not find dormancy in C. tinctoria

as evidenced by a pre-study germination percentage of 94%. Total germination (~90%)

over a 14-day period was equivalent at constant temperatures of 20, 25, or 300C, but the









treatment at 300C had the most rapid rate of germination, reaching approximately 75%

after 2 days. Baskin and Baskin (2001) recommend a 15/250C alternating temperature

regime in light (Baskin and Baskin 2001). Meyer Elliott (1999) broke a mild primary

physiological dormancy after 3 months of dry storage at 250C and achieved 99+0.5%

germination. Achenes remained nondormant after 3 months until the end of testing (7

months) (Meyer Elliott 1999).

Gaillardia Germination and Dormancy Characteristics

The AO SA (1994) protocol for testing achenes of G. pulchella Foug. var. picta

(Sweet) A. Gray is to place achenes on top of a blotter at an alternating 20/300C

temperature plus light during the warm temperature, then recording germination at 4 and

10 days. Light should be provided by a cool-white fluorescent source for a minimum of

8 and a maximum of 16 hr with an intensity of 750 to 1250 lux (75 to 125ft-c), which is

comparable to a fluence rate of 20 Cpmol m-2 SOC-1 or 0.29 to 0.48 mol m-2 day- The light

requirement may be satisfied through the use of one or two 20-W cool white bulbs 15-20

cm above the seeds (AOSA 1994; Baskin and Baskin 2001). Atwater (1980) also tested

G. pulchella var. picta and achieved 70% germination when achenes were tested at a

20/300C alternating temperature for 14 days.

Baskin et al. (1992) conducted a study to examine the effect an alternating wet/dry

storage treatment (simulated natural conditions) would have on dormant G. pulchella

achenes. Storage was performed by placing achenes on a sand substrate in Petri dishes

that were allowed to dry prior to rewetting. Germination tests were conducted by moving

the storage dishes into one of six alternating temperature regimes (6/150C, 10/200C,

15/250C, 15/300C, 20/3 50C, and a simulated Texas regime normal for the given month of

testing). Germination tests were performed under a 14-hr photoperiod for 15 days. After









4 months of storage, dormancy was broken at 10/200C, 15/250C, and 15/280C (simulated

Texas Oct. temperature) resulting in >90 + 3% germination. They also examined achene

burial (stratification) over 4 months in moist sand at five different temperature regimes

(50C, 6/150C, 10/200C, 15/250C, and 15/300C). Germination tests were performed as

above including the simulated Texas temperature regime for October (15/280C).

Dormancy only broke after a 15/250C stratification followed by a germination test at

10/200C (90 + 3%) and after a 15/300C stratification followed by a germination test at

6/150C (100%), 10/200C (100%), or 15/250C (84 + 1%). Gaillardia pulchella~11~11~11~ achenes

exhibited an afterripening type-1 response pattern, which was a first for a winter annual

Asteraceae (Baskin et al. 1992).

Norcini and Aldrich (2003) examined how sowing date affected the establishment

of a Florida ecotype of G. pulchella in northern Florida. Plots (15 ft2) were sown with

2002 harvested achenes during the first week of each month from July through December

2002 and evaluated on March 24, 2003. The largest number of plants was achieved from

the July sowing. The July sowing also visually rated well when judged on flowering,

wildflower density, and weediness.

Rudbeckia Germination and Dormancy Characteristics

The AOSA protocol (1994) for R. hirta requires achenes to be placed on top of a

blotter at an alternating 20/300C in light and germination recorded at 7 and 14 days.

Atwater (1980) tested a Rudbeckia species and achieved 85% achene germination when

tested at a 20/300C alternating temperature for 14 days.

Osmotic priming (osmoconditioning) of Rudbeckia has also been investigated.

Priming in an aerated KNO3 solution osmoticc potential -1.3 MPa) at 300C for 7 days of









a related species, Rudbeckia fulgida Ait., increased the germination percentage and

germination speed as compared to non-primed seeds (Fay 1994).

An early winter (Nov. or Dec.) seeding date is recommended for R. hirta in Florida

so that seeds may be produced by the following summer (Norcini et al. 1999, Pfaff et al.

2002). Norcini and Aldrich (2003) examined how sowing date affected the establishment

of a Florida ecotype of R. hirta in northern Florida. Plots (15 ft2) were sown with 2002

harvested achenes during the first week of each month from July through December 2002

and evaluated on March 24, 2003. The largest number of plants was achieved from the

September sowing. The September sowing also visually rated the best when judged on

flowering, wildflower density, and weediness. Good plant establishment, however, also

occurred during October and November. Sowing of achenes is discouraged from March

through April because of the tendency for dry and/or windy conditions in upland sites

(sandhills) during this time (Pfaff et al. 2002). A thin (7 mm or less) cover of soil is also

recommended after sowing (Norcini et al. 1999).















CHAPTER 2
MATERIALS AND METHODS

Germination characteristics of C. leavenworthii, G. pulchella, and R. hirta, were

determined for freshly harvested achenes that were buried in containers, subj ected to

ambient Florida temperatures, and simulated natural moisture conditions. The mass of all

achenes was estimated by the mean of 10 replicates of 100 achenes.

Coreopsis leavenworthii Origin

Coreopsis leavenworthii achenes were obtained from Melton Farms located near

Dade City (Pasco County, FL). Melton Farms received their achenes from a restoration

site in Polk County, FL, in 2000. The Polk County restoration site was planted with

achenes originating from a site in southwest Orlando (Orange County, FL). Achenes

were harvested on June 19, 2001 and June 19, 2002. Plants at Melton Farms were grown

in rows and provided sub-irrigation once a week. Rows were formed by a 5.1 to 7.6 cm

gap left between two parallel strips of landscape fabric. Achenes were vacuumed off the

fabric and cleaned at Melton Farms. Testing was started on July 4, 2001 and June 24,

2002 for the 2001 and 2002 harvests, respectively.

Gaillardia pulchella Origin

Gaillardia pulchella~11~11~11~ achenes used in all studies were obtained from a first-

generation crop grown at Melton Farms that originated from a wild coastal population in

northern Daytona (Volusia County, FL). Preliminary testing was conducted on naturally

dispersed achenes harvested Julyl3, 2001, August 15, 2001, and October 26, 2001.

Heads of achenes and naturally dispersed achenes were harvested on August 12, 2002 for









the main study. To have enough achenes for the main study, a portion of the achenes had

to be manually removed from their receptacles and combined with achenes that had

naturally dispersed. Testing was started on August 20, 2002.

Rudbeckia hirta Origin

The R. hirta achenes originated from a site in Polk County, FL. Achenes were

produced, harvested, and cleaned at Woodhaven Farms in Havana (Gadsden County, FL)

on August 9, 2002. Testing was started on August 21, 2002.

Species Treatment

Employing methods similar to those suggested by Baskin and Baskin (2001), all

germination testing was initiated within two weeks of harvest.

A small study was conducted in 2001 exploring the effect of time of harvest on

viability and germinability. Replicates (n = 8) of 50 G. pulchella achenes were tested at a

20/300C alternating temperature regime on top of blotter paper with a 12-hr photoperiod

(AOSA 1994). Germination was recorded at 4 and 10 days. On July 13, 2001, in

addition to the methods above, a concurrent test with replicates (n = 3) of 50 achenes was

also run using 0.2% KNO3 in plaCe Of distilled water as the wetting agent.

During the main study, the time of stratification and/or light requirement needed to

break dormancy, the speed of germination, and the change in viability and germinability

as the length of stratification was increased were examined. The C. leavenworthii studies

were approximately one year each for two consecutive years (harvested in 2001 and

2002) while the G. pulchella and R. hirta studies were for one year each (harvested in

2002). Germination treatments were performed each month of stratification (0 to 11

months and 0 to 9 months, respectively, for 2001 and 2002 harvested C. leavenworthii









achenes; 0 to 11 months for G. pulchella and R. hirta). The effect dry-storage had on

viability and germinability approximately 1 year after dispersal was also examined.

In the main study, over 3,200 achenes of C. leavenworthii and G. pulchella were

placed into individual 11 x 14 cm perforated plastic pollination bags for each month of

stratifieation and closed with a drawstring. Due to the smaller achene size of R. hirta,

achenes were placed in 5 x 7 cm sheer cloth envelopes fastened with staples. The C.

leavenworthii, G. pulchella, and R. hirta achenes required for each month of stratifieation

were 0.75 g, 5.00 g, and 1.08 g, respectively. Each bag of achenes was placed on a 7 cm

bed of sand in a standard 3.8-liter black nursery container. Sand was added to containers

until each bag was covered by 7 cm of sand. To prevent the loss of sand through the

drainage holes, each container's exterior was covered with greenhouse ground cloth and

placed into another 3.8-liter container. Each container was initially saturated to Hield

capacity with distilled water and placed on the ground-cloth floor of a plastic-covered

shade house (30% shade). The shade house had retractable plastic sides that maintained

the temperature at the ambient temperature at ground level. The average temperature in

Gainesville, FL, ranges from 7 to 330C (Fig. 2-1). Containers were watered once a week

with 450 ml of distilled water, which is equivalent to 2.5 cm of water per week (Fig. 2-2).

One packet of achenes was removed from its stratifieation container each month and

placed on a pill-counting plate. Masses of achenes were teased apart to aid drying, which

occurred over the subsequent 24 hr. This process was repeated every 3 to 4 weeks. Dry

achenes were counted into 50-seed replicates and placed into 64 test tubes. The 100 x 15

mm plastic Petri dishes had previously been prepared by filling with an average of 62

grams of sand (Standard Sand and Silica Co. 12\20 grit) equivalent to a full, level 30 ml











35

30

^25

~20





-n- Monthly maximum mean
5 Monthly mean

0 -- Monthly minimum mean

J A S O N D J F M A M J
Month


Figure 2-1. Mean minimum, maximum, and monthly temperatures (oC) for Gainesville,
FL, recorded from 1951 through 1980 (SCS 1985).

glass beaker. Each Petri dish of sand was thoroughly moistened with approximately 13

ml distilled water. The 50 achenes were spread as evenly as possible over the moistened

sand. Four dishes per species per incubator were wrapped in plastic film to prevent water

evaporation and another four were covered with one layer of aluminum foil to prevent

light exposure during germination testing. Eight incubators were programmed to

alternate between two temperatures to simulate maximum and minimum daily average

temperature fluctuations in an average Florida year (Fig. 2-1). The eight temperature

regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/330C) were based on

Florida climate data. The 7-W (Bulbrite@ Energy Wiser@ DECO, Moonachie, NJ) soft-

white compact florescent lights were on a 12 hr cycle coinciding with the warmer

temperature of any given regime. Thermostats were accurate to within 20C.











45
40 -ll- Monthly maximum mean
35 -*- Monthly mean
-A- Monthly minimum mean
E 30'


1 5
1" 0





J A S O N D J F M A M J
Month

Figure 2-2. Extreme high, extreme low, and monthly mean precipitation (cm) for
Gainesville, FL, recorded from 1951 through 1980 (SCS 1985).

Germination, defined as radical protrusion >1 mm, was recorded at 7, 14, and 21 d ys

after sowing. Germination of dark-incubated achenes was recorded in a darkroom under

green light (GE 25-W Crystal Color Party! Fiesta! Party Light, Cleveland, OH). After

each 21-day germination count during the testing of the 2002 C. leavemrorthii, G.

pulchella, and R. hirta lots, nongerminated achenes were tested for viability utilizing a

1% 2,3,5 -triphenyltetrazolium chloride (TZ) (Sigma-Aldrich, St. Louis, MO), distilled

water solution. Achenes were removed from their Petri dishes, placed in individual 30 ml

beakers, and covered with the TZ solution. After 24 to 48 hr at 300C in continuous light,

achenes were dissected and classified into four categories: fully red and turgid, partially

red and turgid, white and turgid, and non-turgid or lacking embryo. All turgid achenes

were considered viable due to variability in TZ uptake between treatments. The coolest

germination test temperature (5/200C light), however, was used to evaluate monthly lot

viability in all analysis using a monthly viability correction factor during statistical










analysis. The 5/200C light treatment was utilized because, on average, its viability

deteriorated the least across alternating temperature regimes.

Achenes of the same 2002 C. leavenworthii, G. pulchella, and R. hirta lots were

also dry stored for 14, 12, and 12 months respectively in the climate-controlled laboratory

in open paper bags exposed to florescent lighting. These achenes were tested in an

identical manner to those of all 2002 stratified achenes.

The last germination treatment for the 2001-harvested C. leavenworthii achene lot

was ignored during analysis due to premature harvest of the achene bag from its

stratification container by what appeared to be an animal. Exhumation allowed exposure

of moistened achenes to sunlight. The bag was reburied and germination testing of the

lot was postponed until after all others had been tested to allow for the elimination of

possible error introduction during analysis.

Statistical Analysis

All statistical analyses were performed using SAS (SAS Institute, 2001, Cary, NC).

Germination of viable achenes and fraction of dormant viable achenes were arc-sine

square root transformed prior to analysis in order to normalize the data and stabilize

variances. A three-way ANOVA was performed to evaluate light, month, and

temperature effects and their interactions on viability of achenes, germination of viable

achenes, and dormant achenes. The Bonferroni adjustment was used to diminish the

inflation of type 1 errors caused by the large amount of data.

Scanning Electron Micrograph

Room temperature and humidity equilibrated intact and bisected achenes were

sputter-coated with a gold-palladium alloy through the use of a Denton Vacuum Desk II

sputter coater (Moonachie, NJ). Achene surface morphology and seed coat thickness









images were recorded on a field-emission scanning electron microscope (FESEM,

Hitachi S-4000, Japan) at the Interdisciplinary Center for Biotechnology Research in the

Electron Microscopy Core Laboratory at the University of Florida, Gainesville.















CHAPTER 3
RESULTS

Generally, the main and interactive effects of light, temperature, and months of

stratifieation on germination of viable achenes for all three species were highly

significant (Tables 3-1, 3-2, and 3-3), and especially so for C. leavenworthii (Table 3-1).

Gaillardia pulchella~11~11~11~ had all but one of its interactions significant at the 0.05 level when

germination of viable achene data was examined (Table 3-2). Rudbeckia hirta had Hyve

of its seven interactions significant at the 0.05 level when germination of viable achene

data was examined (Table 3-3).

Table 3-1. Main and interactive effects of temperature, light, and months of stratification
on germination of viable achenes of C. leavenworthii harvested June 19, 2002.

Numerator Denominator
Degrees of Degrees of
Variables Freedom Freedom F Value P Value
Temperature 7 528 49.39 < 0.001
Light 1 528 670.30 < 0.001
Months of stratification 10 528 835.29 < 0.001
Temp. x Light 7 528 34.09 < 0.001
Temp. x Months. 70 528 10.26 < 0.001
Light x Months 10 528 50.12 < 0.001
Temp. x Light x Months 70 528 3.94 < 0.001


2001 Coreopsis leavenworthii

The mean mass of a C. leavenworthii achene was 0. 14 mg.

Due to the lack of post-germination treatment TZ testing and lack of dry storage

testing, only the percent total germination and speed of germination for total achenes










Table 3-2. Main and interactive effects of temperature, light, and months of stratification
on germination of viable achenes of G. pulchella harvested August 12, 2002.


Numerator
Degrees of
Freedom
7
1
12
7
84
12
84


Denominator
Degrees of
Freedom
605
605
605
605
605
605
605


Variables
Temperature
Light
Months of stratification
Temp. x Light
Temp. x Months
Light x Months
Temp. x Light x Months


F Value
58.06
10.29
336.68
4.47
7.82
3.55
1.24


P Value
< 0.001
0.0014
< 0.001
< 0.001
< 0.001
< 0.001
0.0832


Table 3-3. Main and interactive effects of temperature, light, and months of stratification
on germination of viable achenes of R. hirta harvested August 9, 2002.


Numerator
Degrees of
Freedom
7
1
12
7
84
12
84


Denominator
Degrees of
Freedom
624
624
624
624
624
624
624


Variables
Temperature
Light
Months of stratification
Temp. x Light
Temp. x Months
Light x Months
Temp. x Light x Months


F Value
2.16
87.91
249.55
1.52
1.41
7.38
1.23


P Value
0.0356
< 0.001
< 0.001
0.1570
0.0131
< 0.001
0.0910


could be determined for the 2001 harvest. The maximum mean germination for most

treatments occurred early in January 2002 after 6 months of stratification, except for the

10/200C dark treatment where maximum germination occurred one month later (Fig. A-1,

Table 3-4). The maximum mean (+ standard deviation) percent germination for the 16

treatments ran ed from 24.5 + 4.6 % to 77.0 + 5.3 % (Table 3-4 The maximum percent

germination (77.0 + 5.3 %) occurred in the presence of light at 10/200C after 6 months of









stratification, but it was statistically equivalent to 10 other treatments of which most also

occurred after 6 months of stratification (Table 3-4).

A tendency for delayed germination (many achenes taking longer than 7 days to

germinate) was evident at most temperature regimes in both the light and dark for

achenes freshly harvested to those stratified 3 months (Figs. A-2 and A-3). Achene

germination speed at the warmer dark temperatures (15/30, 20/25, 20/30, and 23/330C)

was difficult to determine for most months of testing due to low germination (Figs. A-3E

through A-3H). After 3 or 4 months of stratification (Oct. or Nov.), there was a general

increase in percent germination as of day 7 for all treatments. Germination (between day

0 and 7) continued to increase for most treatments up to 6 months of stratification (Jan.),

at which point almost all reached maximum germination. After 6 months of

stratification, there was a sharp decline until 8 or 9 months of stratification, when

germination was low in all treatments (Fig. A-1). There was another increase in

germination after 8 or 9 months of stratification for most treatments that peaked at 10

months of stratification (Fig. A-1). Many cooler temperature (5/20, 10/20, and 10/250C)

light and dark treatments exhibited increasing delays in germination (from germination

occurring by day 7 to occurring by day 14) from 6 to 10 months of stratification (Figs. A-

2A through A-2C and A-3A through A-3C). After 10 months (Apr.), there was another

sharp decline in germination for all treatments (Fig. A-1).

2002 Coreopsis leavenworthii

The percent germination of viable achenes, the percent total germination, the

speed of germination for total achenes, and the viability of total achenes were examined

for freshly harvested, monthly stratified, and 12 month dry stored achenes from the 2002










Table 3-4. Maximum percent germination (n = 4 + SD) of total 2001-harvested C.
leavenworthii achenes with statistically insignificant months for each
treatment. Achenes were given 21 days of different alternating temperature
regimes under a 12 hr photoperiod or in total darkness. Value in bold is the
overall maximum germination. Values in parentheses are the durations of
stratification not significantly different from the observed maximum within
the same treatment. Months with (*) are not significantly different from
overall observed maximum.

Light


Alternating
temperature
regime (oC)

5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Months of
stratification


6* (10*)
6 (10*)
6* (10)
6*
6
6* (7, 10)
6* (5,10)
6*


Germination (%)

75.5 + 1.7
77.0 + 5.3
70.5 + 4.2
73.5 + 1.9
56.0 + 5.0
70.0 + 4.5
68.0 + 2.9
67.5 + 5.9


Dark


Alternating
temperature
regime (oC)


Months of
stratification


Germination (%)


5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


73.0 + 5.1
64.0 + 2.2
66.0 + 3.9
73.5 + 4.2
24.5 + 4.6
58.5 + 1.0
58.5 + 2.9
39.0 + 1.3


6* (5,10)
7 (10
6 (5 10)

6"

6
6
6


harvest. After 4 months (Oct.) of stratification, germination of viable achenes for all test

conditions was nearly 100% (96.2 + 0.7% to 98.4 + 0. 1%), except for the 23/330C









dark treatment achenes, which increased from 93.3 + 1.6 to 97.8 + 0.2% one month later

(Nov.) (Fig. A-5H). The percent germination of viable achenes remained high ( 87.8 +

1.1%) for the light treatments through the completion of the testing (through 9 months of

stratification). The germination percentages for dark treatments remained high ( 87.1 +

0.8%) for four consecutive months (Oct. through Jan.), then declined. The decline in

germination for the dark-treated achenes was most pronounced at higher temperature

regimes (15/30, 20/25, 20/30, and 23/330C) (Fig. A-5E through Fig. A-5H).

The maximum percent of total germination occurred after 4 to 8 months of

stratification for all light treatments and after 6 to 7 months of stratification for all dark

treatments (Fig. A-4). The maximum percent of total germination for the 16 treatments

ranged from 43.5 + 3.0 to 68.5 + 2. 1 % (Table 3-5). Maximum germination (68.5 + 2. 1

%) occurred at 20/300C light after 6 months of stratification, but was not significantly

different from 10 other treatments (Table 3-5).

Achenes at 5/200C light and dark exhibited delayed germination over more months

of stratification than higher temperature regimes (Fig. A-6A and Fig. A-7A).

Germination at 7 days increased across treatments from fresh harvest through 5 to 7

months of stratification. The most rapid germination was observed after 5 or 6 months

(Nov. or Dec.) of stratification for dark treatments and after 5, 6, or 7 months (Nov.

through Jan.) for light treatments. Light treatments had slower germination over more

months of stratification at cooler temperatures, but germination equivalent to or lower

germination than 9-month stratified treatments also had greater total germination when

compared to dark treatments. After 5 or 6 (Nov. or Dec.) months of stratification there

was a decline in total germination, resulting in higher germination at 7 days of light or









dark as temperature increased. Decreases in germination percentages were greater for

dark treatments compared to respective light treatments. Treatments with the lowest

temperatures (5/200C light and dark) also exhibited a decrease in germination speeds

from 6 and 5 months of stratifieation, respectively, until the end of the experiment (Figs.

A-6 and A-7).

The percentage of viable achenes was influenced by light treatment (Fig. A-8).

For each temperature treatment, those achenes given light generally maintained a higher

percentage of viable achenes over those kept in the dark. The light and dark treatments

generally had percent viabilities ranging from 50 to 65% and 25 to 50%, respectively.

Most treatments exhibited no significant differences between light and dark treatments

when given 7 months of stratifieation.

The treatments stored dry (DS) 14 months (from June 2002 to August 2003) at

room temperature exhibited low percent germination of viable achene (1.1 + 1.1% to 3 8.4

+ 2.3%). All but one DS treatment' s germination was equivalent to or lower than freshly

harvested treatments when given the same temperature and light (Table 3-6). Achenes

DS also exhibited slowed speeds of germination and lower total percent germination

compared to the freshly harvested achenes. The majority of DS treatment viability

ranged between 50 and 65%, similar to those of the stratified treatments exposed to light

(Table 3-7 The neatest overall observed percent viability was 68.7 + 2.6% for the 14-

month DS 15/250C dark treatment, but this was statistically equivalent to a few of the

stratified treatments.

2002 Gaillardia pulchella

The mean mass of a G. pulchella achene was 1.85 mg.











Preliminary testing of G. pulchella achenes harvested in mid-July 2001 revealed


high achene viability and germinability. When tested in accordance with AOSA (1994)

standards, 89.2 + 6.0% of the total achenes germinated (n = 8). The majority of

Table 3-5. Maximum percent germination (n = 4 + SD) of total 2002-harvested C. leavenworthii
achenes with statistically insignificant months for each treatment. Achenes were
given 21 days of different alternating temperature regimes under a 12 hr photoperiod
or in total darkness. Value in bold is the overall maximum germination. Values in
parentheses are the durations of stratification not significantly different from the
observed maximum within the same treatment. Months with (*) are not significantly
different from overall observed maximum.


Light


Alternating
temperature regime
(oC)


5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33



Alternating
temperature regime
(oC)


5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Months of
stratification


Germination (%)


62.5 + 5.5
63.5 + 9.5
64.5 + 3.9
65.0 + 2.4
64.5 + 5.1
65.0 + 2.1
68.5 + 2.1
59.5 + 3.0


7* (1,2,3,4,5,6,9)
3* (2,4,5,6*,7,8,9)
7* (3,5,6)
3* (1,4*,5,7,8)
5* (4,7)
5* (6)
6 (4*)
3 (2)


Dark


Months of
stratification


6* (5,9)
5 (4,7)
5 (4,6)
6 (5)
6 (5)


Germination (%)


65.5 + 6.0
47.5 + 1.3
49.5 + 4.5
53.0 + 2.5
43.5 + 3.0
54.5 + 5.7
47.0 + 1.7
47.0 + 5.1


germination (85.8 + 8.1%) occurred between days 4 and 10. When a concurrent test (n

3) was run using KNO3, 90.7 + 4.6% of all achenes germinated. However, during the










KNO3 treatment, 28.7 + 15.3% of the achenes germinated at day 4 of treatment.

Following two other preliminary germination tests (n = 8 for each) conducted in mid-

August and late October of the same year, only 2.0 + 1.9% of the first and 44.5 + 8.4% of

the second set of achenes germinated. This represented 72.7 + 51.8% and 82.4 + 4.8%

germination of viable achenes, respectively.

Table 3-6. Percent germination (n = 4 + SD) of freshly harvested and 14-month dry
stored (DS) viable C. leavemrorthii achenes harvested in 2002. Achenes were
given a 21-day germination treatment at one of eight different temperature
regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/330C).
Achenes were also exposed to either a 12 hr photoperiod or kept in complete
darkness. Values in bold are the observed maximum germination for the
freshly harvested achenes and for the 14-month dry-stored (DS) treatment.


Light


Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33



Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
germination (%)
71.9 + 1.2
65.4 + 3.1
67.3 + 2.4
68.8 + 1.2
60.7 + 4. 1
57.0 + 5.1
56.5 + 2.9
52.3 + 5.3


DS 14 months
germination (%)
1.1 + 1.1
31.3 + 2.7
24.6 + 2.9
25.4 + 5.7
33.8 + 3.5
32.1 +3.7
33.5 + 3.3
28.5 + 2. 1


Dark


Fresh harvest
germination (%)
47.0 + 3.7
41.5 + 5.4
39.3 + 5.2
48.7 + 4.3
24.9 + 5.1
19.3 + 5.5
11.0 + 2.6
15.3 + 5.2


DS 14 months
germination (%)
2.2 + 1.3
19.9 + 0.6
28.1 + 4.8
38.4 + 2.3
29.5 + 5.0
26.5 + 3.3
16.2 + 4.7
26.4 + 3.5










The percent germination of viable achenes, the percent germination of total

achenes, the percent germination speed of total achenes, and the viability of total achenes

were examined for freshly harvested, monthly stratified, and 12 month dry stored achenes

from the 2002 harvest. The maximum percent germination of total stratified achenes for

each of the 16 treatments occurred within the first 3 months (freshly harvested, one and

two months of stratification) of testing. The maximum percent germination ranged from

28.0 + 5.1 to 42.5 + 4.6% depending on treatment (Table 3-8). Maximum germination

(42.5 + 4.6%) occurred in the 23/330C light treatment after one month of stratifieation

and was not significantly different from 10 other treatments, the maj ority of which also

occurred after 1 month of stratifieation (Table 3-8). Germination of buried achenes also

occurred prior to 1 month of stratifieation and was evident upon harvest. Freshly

harvested, 1, and 2 month-stratified treatments achieved relatively high germination

percentages (approximately 30 to 40%) regardless of temperature and light treatment.

This was followed by a sharp drop in percent germination of total achenes (to

approximately 4%) at 3 months of stratifieation. The drop was followed by a sustained

low (most not achieving > 14%) for the remainder of testing across all 16 treatments.

Freshly harvested G. pulchella achenes yielded viable achene germination from

79.9 + 3.2% to 93.1 + 0.9%. After 1 and 2 months of stratifieation, the percent

Termination ran e narrowed and increased (from 86.6 + 1.8% to 93.1 + 0.8% and from

97.8 + 0.6% to 98.6 + 0.2%, res ectivel ). After 3 months of stratifieation, germination

of viable achenes increased to 100%, decreasing to >92% after 4 months for all 16

treatments. Simultaneously, however, achene viability dropped to between 1 and 18%










during these months and did not increase for the remainder of testing. There was a rapid

decrease in germination of viable achenes for higher temperatures (15/30, 20/25, 20/30,

Table 3-7. Percent viability (n = 4 + SD) of freshly harvested and 14-month dry stored
(DS) C. leavemrorthii achenes harvested in 2002. Achenes were given a 21-
day germination treatment at one of eight different temperature regimes (5/20,
10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/330C). Achenes were also
exposed to either a 12 hr photoperiod or kept in complete darkness. Values in
bold are the observed maximum viability for the freshly harvested achenes
and for the (DS) 14-month dry-stored treatment.


Light


Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
viability (%)
63.0 + 2.8
52.0 + 4.0
54.7 + 3.6
56.7 + 2.3
46.2 + 4.5
41.4 + 2.3
41.0 + 2.6
38.2 + 4.0


DS 14 months
viability (%)
42.7 + 0.5
61.7 + 2.5
56.1 + 2.3
57.4 + 3.9
64.2 + 3.3
62.6 + 3.6
63.8 + 3.2
59.1 +1.8


Dark


Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
viability (%)
33.8 + 2.7
31.0 + 3.3
29.6 + 2.3
35.0 + 2.7
23.7 + 1.5
22.1 + 1.5
20.0 + 1.2
21.0 +1.2


DS 14 months
viability (%)
43.1 + 0.6
52.6 + 0.4
59.4 + 3.8
68.7 + 2.6
60.8 + 4.5
57.7 + 2.8
50.8 + 2.8
57.7 + 2.8


and 23/330C) with a more gradual decline for cooler temperatures (5/20, 10/20, 10/25,

and 15/250C) after 3 months of stratification. Most treatments reached their lowest










percentage germination of viable achenes between 5 and 7 months of stratification

(between January and March). Between 7 and 9 months of stratification, an increase

occurred throughout all treatments in the percent germination of viable achenes. This

increase culminated when all treatments again reached 100% germination of viable

achenes at 11i-months of stratification (July). The second occurrence of maximum

germination of viable achenes (100%), however, was based on less than 16 + 9 of a

possible 100 achenes germinating for any one treatment (Fig. B-1). After having been

DS for 12 months, the germination of viable achenes ranged from 98.3 + 0. 1% to 98.9 +

0.1% for all 16 treatments. Viable achene germination of DS achenes was almost

equivalent to the maximum values set for each stratified treatment and was higher than

observed for freshly harvested achenes (Table 3-9).

Delayed germination was most evident in freshly harvested achenes (Aug.) (Figs.

B-2 and B-3). Freshly harvested low temperature light treatments generally had a slower

speed of germination (Figs. B-2A and B-2B). The speed of germination of DS achenes

was generally more rapid than freshly harvested achenes and was virtually equivalent to

those achenes stratified 1 and 2 months within a given treatment.

The percent viability of total achenes followed the same trend as the percent

germination of total achenes for all 16 treatments. This occurred because most of the

viable achenes germinated each month. During the months when viable achene

germination was the lowest (5 50%), the percent of viable achenes from which

germination could occur was also greatly reduced. There were only negligible differences

between total viability and total germination. The maximum percent viability for each










treatment occurred within the first 3 months of testing and ranged from 28.9 + 2.5% to

46.8 + 4.9%. After 2 months of stratifieation, there was a decline in percent viability

Table 3-8. Maximum percent germination (n = 4 + SD) of total 2002-harvested G.
pulchella achenes with statistically insignificant months for each treatment.
Achenes were given a 21-day germination treatment at one of eight
temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and
23/330C). Achenes were also exposed to a 12 hr photoperiod or kept in
complete darkness. Value in bold is the overall maximum germination.
Values in parentheses are the durations of stratifieation not significantly
different from the observed maximum within the same treatment. Months
with (*) are not significantly different from overall observed maximum.


Light


Alternating
temperature
regime (OC)


Months of
stratification


Germination (%)


5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


39.0 + 2.6
36.0 + 2.2
37.5 + 3.4
36.5 + 3.9
28.0 + 2.7
42.0 + 3.3
38.5 + 3.6
42.5 + 4.6


1* (2*)
2* (1)
1*
1*
2
1* (2*)
1*
1


Dark


Alternating
temperature
regime (oC)

5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Months of
stratification

1*
0* (1,2)
0 (2)
1
1 (0,2)
2 (1)
1
1 (2)


Germination (%)

38.0 + 2.8
36.5 + 6.1
32.0 + 3.7
32.0 + 2.4
30.0 + 3.4
32.0 + 3.7
30.5 + 1.3
28.0 + 5.1










of total achenes (from a maximum of 18.1 + 1.7% to a minimum of 1.0 + 0.0%) for all 16

treatments, with viability remaining depressed for the remainder of testing (Fig. B-4).

The total viability of achenes DS 12 months was almost equivalent to total viability

maximums shown by the stratified achenes when compared within each of the 16

treatments (Table 3-10). Achenes DS 12 months (from Aug. 2002 to Aug. 2003)

generally exhibited equivalent or higher germination percentages than either achenes

freshly harvested or those given any length of stratification (Table 3-11).

2002 Rudbeckia hirta

The mean mass of a R. hirta achene was 0.273 mg.

Percent germination of viable achenes, percent germination of total achenes,

percent germination speed of total achenes, and viability of total achenes were examined

for freshly harvested, monthly stratified, and 12 month dry stored achenes from the 2002

harvest. The maximum percent germination of total achenes for 7 of the 16 treatments

occurred in the freshly harvested achenes (Fig. C-1). The maximum percent germination

of freshly harvested treatments ran ed from 52.0 + 2.4% (23/330C ligt to 12.0 + 2.2%

(20/300C dark). Maximum germination occurred from fresh harvest to the last month of

stratification and ranged from 26.0 + 4.8 (15/250C dark stratified 10 months) to 52.0 +

Table 3-9. Percent germination of viable (n = 4 + SD) freshly harvested and 12-month dry stored (DS) G.
pulchella achenes harvested in 2002. Achenes were given a 21-day germination treatment at
one of eight different temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30,
and 23/330C). Achenes were also exposed to either a 12 hr photoperiod or kept in complete
darkness.



Light
Alternating
temperature Fresh harvest DS 12 months
regime (oC) germination (%) germination (%)
5/20 90.7 + 1.1 98.7 + 0.2
10/20 90.8 + 0.6 98.6 + 0.2










Table 3-9 Continued



Light
Alternating
temperature Fresh harvest DS 12 months
regime (oC) germination (%) germination (%)
10/25 90.5 + 1.5 98.6 + 0.2
15/25 90.7 + 1.6 98.7 + 0.2
15/30 79.9 + 3.2 98.5 + 0.1
20/25 90.4 + 1.5 98.7 + 0.2
20/30 80.4 + 3.4 98.8 + 0.1
23/33 91.6 + 0.8 98.8 + 0.1


Dark
Alternating
temperature Fresh harvest DS 12 months
regime (oC) germination (%) germination (%)
5/20 92.2 + 0.4 98.8 + 0.2
10/20 93.1 + 0.9 98.6 + 0.1
10/25 92.4 + 1.0 98.4 + 0.3
15/25 91.6 + 0.5 98.4 + 0.4
15/30 91.6 + 1.3 98.5 + 0.1
20/25 89.1 + 1.8 98.8 + 0.0
20/30 89.0 + 0.9 98.3 + 0.1
23/33 88.6 + 0.8 98.6 + 0.1

2.4% (23/330C light freshly harvested) (Table 3-12). The freshly harvested 23/330C light

treatment yielding the maximum germination percent (52.0 + 2.4%) was not significantly

different from two other freshly harvested treatments (10/200C light and 15/250C light).

Germination of buried achenes occurred prior to 4 months of stratification and was

evident upon harvest (Fig. 3-1). Percent total germination for all treatments generally










Table 3-10. Percent viability (n = 4 + SD) of freshly harvested and 12-month dry stored
(DS) G. pulchella achenes harvested in 2002. Achenes were given a 21-day
germination treatment at one of eight different temperature regimes (5/20,
10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/330C). Achenes were also
exposed to either a 12 hr photoperiod or kept in complete darkness. Values in
bold are the observed maximum viability for the freshly harvested achenes
and for the 12-month dry-stored (DS) treatment.



Light


Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
viability (%)
27.9 + 3.0
27.6 + 1.9
28.2 + 4.0
29.3 + 4.7
13.3 + 1.7
27.8 + 4.0
14.8 + 3.7
30.8 + 2.9


DS 12 months
viability (%)
39.5 + 4.3
37.3 + 5.8
38.5 + 4.7
40.6 + 6.2
34.4 + 3.1
40.4 + 4.5
42.6 + 2.3
41.1 +3.1


Dark


Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
viability (%)
32.5 + 1.7
38.8 + 6.1
34.5 + 4.2
30.3 + 1.7
31.6 + 4.0
25.1 + 4.0
23.3 + 1.9
22.4 + 1.6


DS 12 months
viability (%)
44.1 + 5.7
35.7 + 2.5
34.2 + 6.1
34.0 + 5.7
33.5 + 3.5
43.6 + 1.9
29.7 + 2.6
36.6 + 3.4


declined after achenes were freshly harvested and reached their lowest after 6 months of

stratification (February). Germination then increased until the end of testing (11 months

of stratification) (July). Freshly harvested achene germination was usually equivalent or

slightly greater than that of achenes stratified 11 months within a treatment. Achenes DS










Table 3-11. Maximum percent germination (n = 4 + SD) of stratified and 12-month dry
stored (DS) G. pulchella achenes harvested in 2002. Achenes were given a
21-day germination treatment at one of eight different temperature regimes
(5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/330C). Achenes were
also exposed to either a 12 hr photoperiod or kept in complete darkness.
Values in bold are the observed maximum germination for the stratified
treatments and for the 12-month dry-stored (DS) treatment.



Light


Alternating
temperature
regime (oC)

5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33



Alternating
temperature
regime (oC)


Months of
stratification

1
2
1
1
2
1
1
1


DS 12 months
germination (%)

39.0 + 3.0
38.0 + 2.4
38.5 + 1.7
40.0 + 3.8
32.5 + 0.8
39.0 + 1.5
42.5 + 1.3
40.5 + 0.0


Germination (%)

39.0 + 2.6
36.0 + 2.2
37.5 + 3.4
36.5 + 3.9
28.0 + 2.7
42.0 + 3.3
38.5 + 3.6
42.5 + 4.6


Dark


Months of DS 12 months
stratification germination (%)


Germination (%)


5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


38.0 + 2.8
36.5 + 6.1
32.0 + 3.7
32.0 + 2.4
30.0 + 3.4
32.0 + 3.7
30.5 + 1.3
28.0 + 5.1


43.0 + 2.2
35.0 + 1.7
33.0 + 1.7
33.5 + 1.4
32.5 + 0.8
39.5 + 2. 1
29.0 + 1.2
35.5 + 1.5


12 months did not germinate as prolifically as either freshly harvested achenes or achenes

stratified 11 months.

Freshly harvested R. hirta achenes had nearly every viable achene germinate (91.6























Figure 3-1. Germinated R. hirta achenes harvested after 4 months of stratification in their
cloth stratification envelope. The bar in the figure is 3.25 cm.

+ 1.8% to 98.1 + 0.1 %) and did not a ain achieve > 90% germination for most

treatments until after 8 months of stratification (Apr.) (Fig. C-1). Achenes given an 8, 10,

and 11 month stratification achieved > 90% germination under most treatments. Viable

Berminatin achenes dro ped to between 6. 1 + 6. 1% and 54.1 + 3.3% when given 6

months stratification (February) across all temperature and light treatments. Viable

achenes given light were usually statistically equivalent to or greater than achenes kept in

the dark within each temperature treatment. Achenes DS 12 months had lower

Termination percent ges (52.7 + 6.2% to 74.6 + 2.4%) than freshly harvested or

treatments stratified for 7 months or more (Table 3-13).

The greatest delay in germination observed for light and dark treatments was at

5/200C and the delay generally decreased as the temperature treatment increased. The

most rapid speed of germination occurred during the first and final month of

stratification, regardless of treatment. Consistent with total germination, germination

speed decreased as stratification progressed toward 6 months of stratification (February)

then the speed increased until testing terminated (July) (Figs. C-2 and C-3). Speed of


:~ rka6 1
~. r
:~T










Table 3-12. Maximum percent germination (n = 4 + SD) of total 2002-harvested R. hirta
achenes with statistically insignificant months for each treatment. Achenes
were given a 21-day germination treatment at one of eight temperature
regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and 23/330C).
Achenes were also exposed to a 12 hr photoperiod or kept in complete
darkness. Value in bold is the overall maximum germination. Values in
parentheses are the durations of stratifieation not significantly different from
the observed maximum within the same treatment. Months with (*) are not
significantly different from overall observed maximum.


Light


Alternating
temperature
regime (OC)


Months of
stratification



0*

0*
2 (0)
2 (11)
2 (1,11)





Months of
stratification




0 (11)
10 (0,11)

0 (11)


Germination (%)


5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


27.5 + 6.2
48.5 + 6.1
34.5 + 1.3
51.5 + 3.3
32.0 + 2.9
33.0 + 2.6
32.5 + 2.4
52.0 + 2.4


Dark


Alternating
temperature
regime (oC)

5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Germination (%)

41.5 + 3.1
37.5 + 2.9
29.5 + 5.4
26.0 + 4.8
35.5 + 6.4
30.0 + 3.2
35.0 + 5.4
42.5 + 4.0


germination for all treatments was generally slower for DS treatments than for either


freshly harvested achenes or those stratified 11 months.







































DS 12 months
germination (%)
61.6 + 4.0
63.1 +1.0
74.6 + 2.4
66.6 + 5.7
56.4 + 3.9
52.7 + 6.2
64.9 + 5.5
68.0 + 2.2


Also, the percentage viable of total achenes for all treatments generally decreased

from fresh harvest to between 5 and 8 months of stratification, then viability increased


until testing terminated (Fig. C-4, Table 3-14).


Table 3-13. Percent germination of viable (n = 4 + SD) freshly harvested and 12-month
dry stored (DS) R. hirta achenes harvested in 2002 for each treatment.
Achenes were given a 21-day germination treatment at one of eight different
temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and
23/330C). Achenes were also exposed to either a 12 hr photoperiod or kept in
complete darkness.


Light


Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
germination (%)
95.9 + 0.9
97.8 + 0.2
96.6 + 0.4
98.0 + 0.1
96.7 + 0.6
96.6 + 0.6
95.6 + 0.4
98.1 + 0.1


DS 12 months
germination (%)
65.6 + 6.8
65.0 + 6.6
73.1 + 5.3
71.6 + 3.2
74.5 + 7.4
70.9 + 4.2
70.9 + 4.0
73.6 + 4.8


Dark


Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
germination (%)
97.7 + 0.1
97.5 + 0.2
96.5 + 0.8
95.9 + 0.7
97.0 + 0.5
96.7 + 0.3
91.6 +1.8
97.8 + 0.2






































Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
viability (%)
45.6 + 2.6
41.7 + 2.8
33.1 + 6.1
27.5 + 4.4
36.4 + 5.7
31.6 + 2.8
13.6 + 2.3
46.6 + 3.4


DS 12 months
viability (%)
25.7 + 3.4
25.7 + 0.7
38.1 + 3.2
32.1 + 7.5
22.2 + 1.8
21.1 + 2.8
29.0 + 4.3
29.9 + 2.0


Table 3-14. Percent viable (n = 4 + SD) of freshly harvested and 12-month dry stored
(DS) viable R. hirta achenes harvested in 2002 for each treatment. Achenes
were given a 21-day germination treatment at one of eight different
temperature regimes (5/20, 10/20, 10/25, 15/25, 15/30, 20/25, 20/30, and
23/330C). Achenes were also exposed to either a 12 hr photoperiod or kept in
complete darkness.


Light


Alternating
temperature
regime (oC)
5/20
10/20
10/25
15/25
15/30
20/25
20/30
23/33


Fresh harvest
viability (%)
27.9 + 5.2
47.8 + 5.4
31.6 + 3.9
51.0 + 3.0
33.3 + 4.5
32.9 + 5.9
23.7 + 2.3
53.6 + 2.5


DS 12 months
viability (%)
30.4 + 4.9
30.2 + 5.8
38.6 + 6.1
34.5 + 3.5
44.2 + 8.3
34.9 + 5.6
34.9 + 5.8
38.9 + 5.7


Dark















CHAPTER 4
DISCUSSION AND CONCLUSIONS

Coreopsis leavenworthii

If stratified under ambient Florida temperatures, primary physiological conditional

dormancy of commercially produced north central Florida C. leavemrorthii achenes

harvested in mid-June is broken between the end of October and the beginning of January

with a type-1 response pattern. The 2002 harvest' s percent germination of viable

stratified achenes ranged from 93.3 + 1.6% to 98.4 + 0.1% during this time over a wide

range of temperature regimes in both the light and dark. High total achene germination

and an increase in the speed of germination (large proportion of achenes germinating

prior to 7 days of treatment) during both years of study also supported this conclusion.

The last germination treatment for the 2001-harvested achene lot (6/4/02) was

ignored during analysis due to premature harvest of the achene bag from its stratifieation

container, thus allowing exposure of moistened achenes to sunlight. A phytochrome

response is hypothesized to have initiated germination upon reburial (Bewley and Black

1994). The depth of burial (7 cm) and limited achene embryo energy stores would have

proven a lethal combination if germination were to have occurred prior to proper harvest

(Bewley and Black 1994). It could not be assumed that the resulting Einal months' low

germination was independent of the premature sunlight exposure; so all treatments from

this length of stratifieation were eliminated from further scrutiny. Most of the more

informative aspects of the 2001 and 2002 harvests could not be compared to each other









directly because of the lack of TZ testing in the 2001 harvest. The only comparisons that

were made were from total germination and speed of germination.

During both years of testing, soon after harvest (mid-June) many C. leavemrorthii

achenes appeared to be conditionally dormant. Dormancy was evident from slow

germination speeds (most viable achenes requiring more than 7 days to germinate) during

both years and suppressed viable achene germination (ranging from 25 to 89% dormant,

depending on treatment) of the 2002 harvest. Freshly harvested achenes were most

responsive when germinated in the light at cooler temperatures and they were least

responsive when germinated in the dark at warmer temperatures. For the 2002 harvest,

achenes broke conditional dormancy after 4 months of stratification for all treatments.

During the months leading up to conditional dormancy break, achenes gradually

germinated to increasingly higher percentages at warmer temperatures. This indicated

that conditional dormancy was fully broken by October exhibiting a type-1 response

pattern. From the time achenes became nondormant until the study ended, viable achenes

would germinate as long as they were provided with light. Germination of viable

achenes decreased after 5 or 6 months of stratification when achenes were not provided

light indicating a secondary conditional dormancy; this trend continued until testing

ended. This decrease in germination and secondary conditional dormancy onset was

hastened in the dark treatments as germination temperature regimes increased. Also, as

temperature regimes increased in the dark treatments, there was a reduction in achene

viability. Reduced viability may have led to an exaggeration in the decrease observed in

viable achene germination, which might have occurred because the basis for determining









the decline in dark-treatment viable-achene germination is intimately associated with

total achene viability.

Dark achenes appeared to enter a secondary conditional dormancy after

approximately 6 or 7 months of stratification. However, the relationship between total lot

viability and high temperature dark achene deterioration makes this deduction somewhat

speculative. Achenes exposed to light, on the other hand, are believed to have shown a

phytochrome response, thus allowing viable achene germination to remain high after

conditional dormancy was broken. Not only did light improve germination, it also

maintained viability over the dark treatments each month after they became nondormant.

This resulted in a larger number of viable achenes from which germination could occur.

Darkness also significantly degraded lot viability, thus reducing the germination

potential. After conditional dormancy break, viability appeared to be negatively affected

by an increase in germination temperature and the absence of light within each month of

stratification. As stratification duration lengthened after achenes became nondormant, it,

too, negatively affected achene viability.

The environmental parameters (moisture requirements, light exposure usually

found growing, etc.) of the species and the state of the seed at dispersal (dormancy,

viability, light requirements, favorable germination temperature) should be taken into

account when deciding on an area to sow seed. As seen from this study, a portion of mid-

June harvested C. leavemrorthii achenes are conditionally dormant upon dispersal when

commercially produced in north central peninsular Florida. The most expedient way for

the achenes to break conditional dormancy when dispersed into a natural setting would

appear to be 4 to 6 months of stratification (until October to early January if dispersed in










June). In the native habitat a stratification of this type might occur if achenes were buried

(entered into the seed bank) to a depth greater than that of sunlight penetration

(approximately 2-10 mm depending on the soil particle size, color, and moisture content)

(Baskin and Baskin 2001). Then, after 4 to 6 months, the achenes would ideally be

brought to the surface to a depth where they could receive exposure to light but still be

buried enough so the surrounding soil could provide sufficient moisture to break

quiescence. The moist habitats (wet pine flatwoods, glades, prairies, and ruderal wet

ditches) in which C. leavenworthii is commonly found should be able to provide the

water necessary for germination and sustained growth (Ledin 1951; Taylor 1998).

Conditional dormancy should remain broken for at least one year from dispersal and

possibly beyond provided light continues to be available to the achenes. During an

average December in Gainesville, Florida (the same time C. leavenworthii achenes

became nondormant), the mean high and low temperature would typically be between 21

and 80C, respectively (SCS 1985). This cool December temperature is very similar to the

5/200C or 10/200C treatments, which usually retained high viability and also usually had

a large percentage of viable achenes germinate as compared to other temperature regimes

over the year of stratification. The data supports a late fall to early winter sowing if

achenes are nondormant due to a proper pretreatment to break the achene's conditional

dormancy. Evidence of successful winter seeding has proven to be more reliable in some

species than those performed in the summer despite the lack of dependable moisture

(Pfaff et al. 2002). This is believed to occur because of cooler soil temperatures or a

reduction in weed competition during this time of year. However, sowing achenes soon

after harvest (summer to fall) in a low moist area and sowing deep enough to prevent










light exposure (> 1 cm) may act as an effective pretreatment/stratification. The natural

dormancy breaking process may need to be artificially streamlined by timing procedures

in order to achieve a uniform C. leavenworthii stand. A consistently moist environment

similar to that provided in this thesis may be crucial in overcoming dormancy within 4 to

6 months. A mid-October tilling to the depth of the achenes would bring achenes to the

surface, exposing them to light and would remove them from a possible anoxic

environment while still retaining a moist environment. This procedure would make an

attempt at approximating experimental procedures that effectively overcame C.

leavenworthii's primary conditional dormancy 93.3 + 1.6% to 98.4 + 0. 1% germination

of viable achenes). This however may or may not be practical for FDOT procedures.


Without light, viability tends to be lost and the chance of the achenes entering a

secondary conditional dormancy increases, especially when temperature increases nearer

to 330C. Achenes could experience this situation if they were to remain close enough to

the soil's surface to experience solar heating, but were buried deep enough to prevent

sunlight exposure. This translates into reduced viability and the implementation of a

secondary conditional dormancy in achenes that remain buried in the seed bank during

moist summer months of the following year (May through Sept.). Achenes probably

experience a phytochrome response after 4 months of stratification to delay secondary

conditional dormancy onset as long as favorable germination conditions are present. The

steadily lengthening of time required for germination to occur, observed at some of the

cooler temperature regimes when C. leavenworthii germination speed was examined,

would protect achenes from germinating unless there was a significantly prolonged

period of favorable germination conditions.









For C. leavenworthii achenes, the effect of dry storage (DS) for approximately one

year from harvest remains unresolved. Because of limited testing, it is unclear whether

conditional dormancy was broken prior to 14 months of dry storage. It is evident,

however, that 14 months of dry storage is an ineffective dormancy-breaking treatment.

Germination of dry-stored achenes was negligible (62 to 99% remained dormant) when

compared to that of any stratified treatment. In many instances, DS treatments developed

a deeper dormancy when compared to that of any stratification length of a similar

treatment. The increased dormancy was recognizable from the large number of viable

achenes that did not germinate and from slow germination. Dry storage is, however,

effective at maintaining achene viability for more than one year. If C. leavenworthii

achenes are stored for 14 months then a dormancy breaking treatment will likely need to

be employed prior to sowing in order for germination to occur.

While C. tinctoria is most closely related phylogenetically to C. leavenworthii and

both species occupy wet, open canopy sites, their germination requirements do not seem

to have many similarities (Kim et al. 1999; Wunderlin and Hansen 2003). The AOSA

(1994) suggests a constant 200C germination environment for C. tinctoria. Kaspar and

McWilliams (1982) demonstrated that an illuminated constant 20, 25, or 300C

environment yielded favorable germination and achenes did not exhibit dormancy during

testing. Conversely, Baskin and Baskin (2001) concluded that an illuminated, alternating

15/250C temperature regime provided optimal germination. However, similar to that of

C. leavenworthii, Meyer Elliott (1999) found a mild primary physiological dormancy in

C. tinctoria. Meyer Elliott (1999) broke dormancy in C. tinctoria after 3 months of dry

storage at 250C and dormancy remained broken for the remainder of testing (7 months).









Coreopsis leavenworthii, however, did not respond to a 14-month dry storage. Primary

physiological conditional dormancy in C. leavenworthii was only broken when stratified

and it responded favorably to a combination of alternating cool temperature (5/200C) and

12 hr illumination.

One foreseeable obstacle that may be encountered during attempted cultivation of

C. leavenworthii outside of its native habitat would stem from insufficient moisture

required to break quiescence and allow germination. Judging by its natural habitat (wet

pine flatwoods, glades, prairies, and ruderal wet ditches), it may be advisable to sow

achenes in moist ditches, depressions, and poorly drained areas that have evidence of

standing water (Ledin 1951; Taylor 1998). Soil texture should also be examined prior to

sowing (Pfaff et al. 2002). Another possible explanation for poor stand establishment

would be the sowing of conditionally dormant achenes. Dormancy as a factor

influencing desired stand establishment may be overcome by a simulated moist

stratification of freshly harvested achenes for 4 to 6 months, but another option may be

the implementation of a chemical pretreatment. Some chemicals can take the place of or

reduce the need for light and afterripening in Asteraceae are ethylene and, more

commonly, gibberellic acid (commonly applied as GA3, GA4, Or GA7) (Bewley and

Black 1994).

Gaillardia pulchella

Gaillardia pulchella~11~11~11~ achenes harvested in late summer (mid-August) appeared to

have a very weak primary physiological conditional dormancy; however, they could be

considered nondormant (due to a germination of viable achenes of approximately 90% at

almost all treatments. Slow germination (many achenes taking 14 to 21 days to










germinate) and a slightly below total germination of viable achenes characterized the

mild conditional dormancy.

Prior to 1 month of stratifieation, many achenes buried in the harvested container

had germinated. This event and the subsequent removal of germinated achenes prior to

germination testing resulted in a reduction of that month' s total viability of the achenes

tested. If these achenes had not germinated in their stratifieation container and were able

to be included in germination testing, a higher percent viability would have been

observed and a higher germination of viable achenes may have been observed. After 2

months of stratifieation, achene viability and the total amount of achenes that germinated

declined, but the percentage of viable germinating achenes increased and remained high

for one additional month until the fourth month of stratifieation, when it decreased

slightly. From this, it may be assumed that buried achenes germinated in all stratifieation

containers prior to 3 months of stratification (mid-November). Strati fi cati on-container

germination would explain the abrupt decline in viability and germinability experienced

from mid-November until the end of testing.

The mild conditional dormancy of the late summer-harvested achenes broke

between fresh harvest and 3 months of stratification, but full dormancy break (all

treatments exhibiting 100% germination of viable achenes), at 3 months of stratification,

was only based on a low number of viable achenes. So, when achenes are stratified, a

sharp loss of viability may be expected following dormancy break if emergence is not

achieved.

A limited number of achenes remain dormant after mild primary conditional

dormancy breaks. These achenes appeared to enter a secondary conditional dormancy









when stratification continued. The majority of those achenes go into dormancy during

the cooler months of the year (initiating prior to January) and dormancy continues to

deepen as the environmental temperature decreases (through winter). This strategy

would allow a few of the achenes that have entered the seed bank to germinate (break

dormancy) only after a prolonged time (possibly the next season). As is evident from the

study, the maj ority of surviving viable achenes reached their deepest conditional

dormancy within 7 months of stratification at the cooler temperatures (5/20, 10/20, and

10/25, plus 15/250C dark). At warmer temperatures (15/25 light, plus 15/30, 20/25,

20/30, and 23/330C) achenes became deeply dormant sooner (between 5 and 6 months of

stratification). This separation in time between cool and warm dormancy onset would

allow achene germination if exhumation were to occur or if the achenes were not buried

excessively deep when temperatures were cool enough to support successful seedling

establishment. During the month of January, the temperature in Gainesville, Florida,

which averages 7 to 190C, would be cool enough to delay some of the remaining viable

achenes' dormancy onset until March when the temperature would be warmer (averaging

11 to 240C in Gainesville, Florida) (Fig. 2-1) (SCS 1985). This temporal separation

would also cause surviving achenes to become dormant and remain in the seed bank

during an unseasonably warm winter.

Across all treatments, the deepest dormancy was followed by another break in

dormancy (all treatments again exhibiting 100% germination of viable achenes), which

occurred after 11 months of stratification (mid-July). Secondary conditional dormancy

appeared to be broken during mid-summer when temperatures are at their warmest










(averaging 22 to 330C). The observation of dormancy and its subsequent break was,

however, based on a greatly reduced number of viable achenes.

The behavior of DS G. pulchella achenes from the late summer (mid-August)

harvest remains unresolved due to limited testing. However, after 12 months of dry

storage, achenes were nondormant and they had retained a high level of viability. When

DS treatments were compared to stratified treatments, the DS treatments performed as

well or better than stratified treatments (equivalent or higher germination of viable

achenes, total achene germination, and speed of germination). It appears that dry storage

(afterripening) of G. pulchella for approximately one year from harvest would be easier

and possibly more effective at breaking dormancy, maintaining viability, and maintaining

an ease of implementation for roadside establishment than stratification would be. A

plant date one year from harvest of the achene lot studied would be in the middle of

August the following year. August in Gainesville and throughout the rest of Florida is,

however, the hottest time of year (averaging 22 to 330C) (SCS 1985). For those trying to

germinate achenes in nonirrigated environments, sowing in August might increase the

need for supplemental water. Dry storage durations of greater than or less than a year

from harvest may also provide similar results, but this would need to be examined in

future studies. Ideally, the achenes would be sown just prior to the season of most

dependable moisture (the rainy season usually begins in June) to alleviate the need for

externally applied moisture (Pfaff et al. 2002). The months in which the most

precipitation occurs, however, are also the warmest months of the year.

Freshly-harvested, viable achenes have the ability to germinate in high percentages,

so freshly harvested achenes could be sown if time is of utmost importance. This course









of action would require external care for a longer duration (for example, at least two

weeks of supplemental irrigation) due to the slow speed of germination found in fresh

achenes. A slightly lower proportion of viable achene germination would also be

expected. Light does not seem to play a crucial role in the germination process and only

negligible differences between light and dark treatments were observed in viable achene

germination. This would allow achenes to be sown under a light cover of soil, which

would provide for better moisture retention around the achenes. Gaillardia pulchella~11~11~11~

achenes do not have a large amount of stored energy due to their small size and should

most likely not be buried too deeply. Problems currently experienced with stand

establishment are most likely related to water requirements of achenes prior to

germination and throughout seedling establishment. Sufficient water is critical if

dormancy has not been fully broken.

Many biotic and abiotic factors can affect viability. As seen from preliminary

testing, the viability of different harvests during various months is highly variable. An

unsatisfactory stand may result not from the treatment provided, but from poor initial

viability. A preliminary TZ test in accordance with AOSA (1994) standards or simply

pinching a small sample of imbibed achenes with tweezers in order to find turgid

embryos would provide valuable information when determining seeding rate prior to

sowing. During preliminary testing, a 2001 mid-July harvest exhibited almost 90%

germination. This is compelling evidence, although it does not prove conclusively that

July is the ideal time to harvest. A preliminary 2001 mid-August harvest had only 2.8%

viability out of the 400 achenes tested, but a mid-August harvest the following year (the

2002 lot studied in detail) had a fresh harvest viability average of 27.4% when 3200









achenes were put through variable temperature regime germination testing prior to

viability testing. Evidently, there may be a great deal of viability variation among

months within a year and between successive years, so viability testing is critical before

the purchase of any lot of achenes.

Preliminary testing also hinted that although a large number of achenes may not

appear to have germinated, the amount of germinating viable achenes may have been

substantially high. A viable achene germination of 72.7% (mid-August), 82.4% (late

October), > 89.2% (mid-July, value taken from germination of total not germination of

viable achenes), and 89.5% (mean from the fresh achene viability of the 2002 lot studied

in detail) seems to demonstrate that, regardless of lot viability, a large proportion of

freshly harvested viable G. pulchella achenes can be expected to germinate when

favorable conditions are present.

Rudbeckia hirta

Most R. hirta achenes appear non-dormant upon fresh harvest in late summer (mid-

Au ust). This is evident from hi h viable achene germination (1 95%) in all but one

treatment (20/300C dark). Most freshly harvested achenes initiated germination prior to 7

days of treatment in all but the coolest treatments (5/200C light and dark treatments).

The maj ority of germination in both 5/200C treatments occurred between 7 and 14 days.

Low temperatures are believed to significantly affect the physiological mechanisms

responsible for germination through slowed enzyme reactions resulting in sluggish

germination (Bewley and Black 1994). As previously stated, the literature recommends

sowing R. hirta achenes in the late fall or early winter (Sept. through Dec.) so achenes

may be produced by the following summer (Norcini and Aldrich 2003, Norcini et al.










1999, Pfaff et al. 2002). Over the following 6 months of stratifieation, achenes on

average gradually entered a physiological conditional dormancy. The progression into

dormancy was characterized by lowered germination of viable achenes and lowered

germination of total achenes and from a slowed speed of achene germination. After 4

months of stratifieation, though, achene germination prior to harvest occurred in the

stratifieation container. As seen for G. pulchella, this event is believed to have reduced

the initial viability and observed germination due to the removal of pre-germinated

achenes prior to germination testing. Pre-germinated achenes were subsequently not

factored into the fourth month' s germination totals. This removal of a number of non-

dormant, viable achenes may have caused the depression in germination of viable

achenes for that month.

The only significant dormancy incurred was at 6 months of stratifieation (February)

when the germination of viable achenes ra ged from 6.1 + 6.1 to 54.1 + 3.3%.

Conditional dormancy broke 5 months later (July) and was characterized by an increased

germination speed achenee germination prior to 7 days of treatment), greater viable

achene germination (from 88.9 + 3.3 to 93.4 + 0.5% and heater total achene

germination during all test treatments.

The erratic nature of the R. hirta germination of viable achene results may stem

from the way in which this value is calculated. This point can best be demonstrated by an

exaggerated example. When the only viable achene germinates during a germination test

100% of viable achenes germinated. The previous situation appears equivalent to a

germination test where 200 viable achenes germinated which would also appear as 100%

germination of viable achenes. So, with a low initial viability a difference of just a few









germinating or dormant viable achenes can affect the calculated percentage of

germinating viable achenes greatly. The lack of pregermination testing of viability may

have also affected the calculations due to possible viability determination errors. This is

discussed further in Triphenyltetrazolium Chloride Testing Technique section at the end

of this chapter.

The behavior of DS R. hirta achenes from a late summer (mid-August) harvest to

approximately one year later remains unresolved. It could not be determined if

conditional dormancy became deeper or if it was fully overcome during the prior 1 1

months of storage due to limited testing. After 12 months of dry storage, though, all

achenes exposed to the various germination treatments showed signs of having developed

a deeper dormancy over freshly harvested or 11i-month stratified achenes. For most

treatments, this was evident from lower germination of viable achenes and was reinforced

by essentially the same viability as freshly harvested or 11i-month stratified achenes. Dry

storage evidently is not an effective dormancy breaking treatment, but it can maintain

viability.

Across all treatments, there was an unexpected, gradual dip and subsequent rise in

total viability during the course of testing. One possible explanation for this phenomenon

is that dormant achenes exposed to the rigors of the germination process have a hampered

ability to retain viability as compared to achenes that delay germination until they are in a

state of non-dormancy. The need for pre-treatment viability (TZ) testing is important in

any future study of this species because achene viability appears to be greatly affected by

germination testing. Banyai and Barabas (2002) state that viability and expected









germination should be equivalent as long as dormancy is not present and as long as a seed

lot has not been deteriorated from a germination test of normal or extended duration.

Triphenyltetrazolium Chloride Testing Technique

There should be no significant difference among the total viability of subsamples of

a particular seed lot when total percent viable seed is calculated as percent germinated

seed plus dormant seed (based on a post germination TZ test) as long as the germination

test itself does not reduce viability. However, this was not the case in our work, and

highlights the need for additional research about TZ testing standards. The coolest

germination test temperature (5/200C light) was used to evaluate monthly viability during

statistical analysis through the employment of a monthly viability correction factor. The

5/200C light treatment was used because the viability of achenes in this germination test

regime deteriorated the least on average across monthly stratification treatments.

Because each of the three species tested had some degree of dormancy and because of

reduced monthly lot viability associated with an increase in germination treatment

temperatures, viability was assigned to turgid achenes instead of those testing positive in

the post-germination TZ tests in order to reduce false negatives (Baskin and Baskin 2001;

J.G. Norcini personal communication). The determination of viability from turgid

embryos after a germination test, however, still does not appear to be able to determine

initial pregermination testing viability. Because seeds have been known to give faulty TZ

results if dormant and/or deteriorated from a germination test, it would be advisable in

future studies involving these three species to perform a pre-germination TZ test to each

stratified lot upon harvest and post-germination TZ tests on all nongerminated achenes.

This procedure should help to minimize and quantify the effect of germination test









deterioration. It would also provide more insight into the effect that temperature and light

treatments had on viability. It is also advisable to track turgid embryos in order to

eliminate the possibility of TZ testing error as a result of a previously unknown dormancy

in an unstudied species. In a study of this nature, dormancy may be anticipated, but not

avoided, as a possible factor influencing TZ testing.

Implications for Roadside Plantings

Coreopsis leavenworthii (mid-June Harvest)

55 to 70% of freshly harvested viable achenes may be able to germinate if

sown close enough to the soil surface to receive light and provided

sufficient moisture is available

Sow shallowly (< 7 mm), due to limited energy storage, and for needed

moisture retention (Baskin and Baskin 2001, Norcini et al. 1999)

Sow in low areas due to species moist habitat background and to reduce the

need for supplemental water

Dormancy may be lost 4 to 6 months after sowing if dark stratified at

equivalent Gainesville, Florida temperatures

If dark stratification continues and germination does not occur, achenes tend

to enter a secondary dormancy

In drier sites, water thoroughly for at least 2 weeks to facilitate germination

Mow in the late fall after achene development to disperse mature achenes

A 14-month dry storage for can maintain viability









Gaillardia pulchella (Late Season Harvest)

Achenes stored dry in a climate controlled facility for 1 year will germinate

more rapidly and uniformly than freshly harvested achenes

Mild primary dormancy is broken prior to 1 month when stratified

A few achenes (I 10%) can be expected to enter a secondary dormancy if

stratified

Sow shallowly (I 1 cm) due to limited energy storage and for needed

moisture retention (Baskin and Baskin 2001, Norcini et al. 1999)

Sow in the early fall

If sowing one year dry stored achenes, keep soil moist for at least 1 week

If sowing freshly harvested achenes, keep soil moist for at least 2 weeks due

to slowed germination of approximately half the achenes able to germinate

Mow in the late fall after achene development to disperse mature achenes

A 12-month dry storage can maintain viability

Rudbeckia hirta (Late Season Harvest)

Physiological dormancy seems to develop soon after maturation when

achenes are stratified

Dormancy is deepest 6 months after maturation when stratified and can be

expected to take one year to become nondormant

Sow freshly harvested achenes shallowly (< 7 mm) due to limited energy

storage, needed moisture retention, and to provide light to the achene

(Baskin and Baskin 2001, Norcini et al. 1999)

Sow in the fall









* Sow freshly harvested achenes and keep soil moist for at least 2 weeks due

to slowed germination of up to half the achenes able to germinate

* Mow in the late fall after achene development to disperse mature achenes

* A 12-month dry storage only degrades viability minimally














APPENDIX A
Coreopsis learc~11in 1, thrii FIGURES


90
80
70

6 0

- 40

S30


7/4 8/3 9/2 10/2 11/1 12/1 12/31 1/30 3/1 3/31 4/30 5/30

Day germination treatment initiated (30 day scale)

Figure A-1. Mean (n = 4) percent germination (+ SD) of total 2001-harvested C.
leavenworthii achenes. Achenes were subjected to a 21-day germination
treatment for both a 12 hr photoperiod and in complete darkness at each of
eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F.
20/25, G. 20/30, and H. 23/330C) over 11 months of stratification.










90

80 -H *-10/200C LightB

70 -H .-10/200C Dark


0 50

-"40



10'

10

7/4 8/3 9/2 10/2 1 1/1 12/1 12/31 1/30 3/1 3/3 1 4/3 0 5/30

Day germination treatment initiated (30 day scale)


90

80 -1 10/250C Light

70 -H --10/250C Dark


0 50

--40




20


7/4 8/3 9/2 10/2 1 1/1 12/1 12/31 1/30 3/1 3/3 1 4/3 0 5/30

Day germination treatment initiated (30 day scale)

Figure A-1. Continued



















































7/4 8/3 9/2 10/2 1 1/1 12/1 12/31 1/30 3/1 3/3 1 4/3 0 5/30

Day germination treatment initiated (30 day scale)

Figure A-1. Continued


- --15/250C LightD

---.15/250C Dark


r/4 8/3 9/2 10/2 1 1/1 12/1 12/31 1/30 3/1 3/3 1 4/3 0 5/30

Day germination treatment initiated (30 day scale)




-* 15/300"C LightE

.1 5/30"C Dark


90

80

70


60


0 5



10

O






90

80

5

7 0

6 0

.9 0




10
























































Figure A-1. Continued


G
- 20/300"C Light

- 20/300"C Dark


90

80 -H *-20/250C Light

---1 20/250C Dark


0 50

40


S0.
10'

10

7/4 8/3 9/2 10/2 1 1/1 12/1 12/31 1/30 3/1 3/3 1 4/3 0 5/30

Day germination treatment initiated (30 day scale)


90

80

70


0 50

-40


20


10


7/4 8/3 9/2 10/2 1 1/1 12/1 12/31 1/30 3/1 3/3 1 4/3 0 5/30

Day germination treatment initiated (30 day scale)











H
-*-23/330C Light

- 23/330C Dark


70


0 50

40


7/4 8/3 9/2 10/2 1 1/1 12/1 12/31 1/30 3/1 3/3 1 4/30o 5/30

Day germination treatment initiated (30 day scale)

Figure A-1. Continued
90

80 -- 0 7 Day

70 O 14 Day

7o 60 ~--1 21 Day

.0 50 -

.40

S30 1
20




J A S O N D J F M A M J

Month

Figure A-2. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2001-
harvested C. leavenworthii achenes at a 12 hr photoperiod. Achenes were
subj ected to a 21-day germination treatment, at each of eight temperature
regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30,
and H. 23/330C) over 11 months of stratification.



















































N D J F M
Month


--07 Day

- O 14 Day

- M21 Day


90

80

70


0 50

40


20

01

0






90

80

70


0 50

-40


20

1 0


A


S


O


A M


J


Figure A-2. Continued


J A S O ND J F M AM J

Month










90

80

70


0 50

40


20

10
0






90

80

70


0 50

40


20

10
0


J A S O ND J F M AM J
Month


J A S O ND J F M AM J
Month


Figure A-2. Continued












S07 Day

SO 14 Day

21 Day











J A S O ND J F M AM J

Month


90

80

70


0 50

40


20

10

0


Figure A-2. Continued


90

80 -

70 -


0 50

.40


20

10 -


J A S O ND J F M AM J

Month














































Month

Figure A-3. Mean (n = 4) percent germination (+ SD) at 7, 14, and 21 days for the 2001-
harvested C. leavenworthii achenes in complete darkness. Achenes were
subj ected to a 21-day germination treatment, at each of eight temperature
regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F. 20/25, G. 20/30,
and H. 23/330C) over 11 months of stratification.


-1~


,PL I


S07 Day
SO 14 Day
21 Day


J A S O N D J F M AM J
Month

A-2. Continued


- fu r. & r. F


90
80
70


0 50
40


20
10


i~


~R, ~- P~L


I


-
-
-


Figure ,
90
80
70



.40
~30
20

10


S07 Day
SO 14 Day
21 Day


J F M










90

80

70


0 50

.40


20

10

0






90

80

70


0 50

.40


20

1 0

O


J A S O ND J F M AM J

Month


S07 Day

SO 14 Day

- 521 Day


rk...


J A S O N D


F M AM J


Month


Figure A-3. Continued


I











90

80

70



60


050

. 0

-



30


20



10
-

20




90

-


J A S O ND J F M AM J

Month


S07 Day

SO 14 Day

- 521 Day


A S O N D


F M AM J


Month


Figure A-3. Continued











- 0 7 Day
- O 14 Day
-- 21 Day









J A S O ND J F M AM J


90
80
70


0 50
40


20
10
0


Month


90
80
70


0 50
. 40
-

20
10
-


S07 Day
SO 14 Day
21 Day


A S O N D J F M A M J


Month


Figure A-3. Continued


P~iR~T


R,










90

80 -- 07 Day

70 O 14 Day

60 -- 21 Day


40

S30
20
10


J A S O N D J F M A M J
Month

Figure A-3. Continued


50

Ei 40

S30

20

10


6/24


7/24 8/23


9/22 10/22 11/21 12/21 1/20 2/19 3/21


Day germination treatment initiated (30 day scale)

Figure A-4. Mean (n = 4) percent germination (+ SD) of total 2002-harvested C.
leavenworthii achenes. Achenes were subjected to a 21-day germination
treatment for both a 12 hr photoperiod and in complete darkness at each of
eight temperature regimes (A. 5/20, B. 10/20, C. 10/25, D. 15/25, E. 15/30, F.
20/25, G. 20/30, and H. 23/330C) over 9 months of stratification.






















































Figure A-4. Continued


-*-10/20C Light B
---10/200C Dark


6/24 7/24 8/23 9/22 10/22 11/21 12/21 1/20 2/19 3/:

Day germination treatment initiated (30 day scale)


10

0


21


6/24


7/24 8/23 9/22 10/22 11/21 12/21 1/20 2/19 3/21

Day germination treatment initiated (30 day scale)

































-* 1 5/300"C Light E
-- 1 5/300"C Dark


S50

fi 40

S30


6/24 7/24 8/23


9/22 10/22 11/21 12/21 1/20 2/19


3/21


Day germination treatment initiated (30 day scale)


6/24 7/24 8/23


9/22 10/22 11/21 12/21 1/20 2/19


3/21


Day germination treatment initiated (30 day scale)


Figure A-4. Continued






















































Figure A-4. Continued


-* 20/250C Light F
-- 20/250C Dark


/24


20

10

0
6


7/24 8/23 9/22 10/22 11/21 12/21 1/20

Day germination treatment initiated (30 day scale)


2/19


3/21


6/24


7/24 8/23 9/22 10/22 11/21 12/21 1/20 2/19 3/21

Day germination treatment initiated (30 day scale)